Estrogen Receptor 1 Mutations Research Articles

Abstract PO4-27-05: A phase I open-label dose escalation trial of FWD1802 as monotherapy and in combination with palbociclib in patients with ER+/HER2- unresectable locally advanced or metastatic breast cancer with or without ESR1 mutations

Abstract Background: Estrogen receptor (ER) signaling sits at center of the tumor biology in ER+/HER2- breast cancers. Current standard of care involves hormone therapy with agents including selective estrogen receptor modulators (SERMs), selective estrogen receptor degraders (SERDs), and aromatase inhibitors (AIs). Unfortunately, most patients eventually develop resistance to these therapies. Mutations in estrogen receptor 1 (ESR1) are partially responsible for the acquired resistance. Mutated estrogen receptors are still sensitive to fulvestrant but with a much less sensitivity. In January 2023, elacestrant received its first approval for the treatment of postmenopausal women or adult men with breast cancer harboring ESR1 mutations in US. However, there is still great unmet medical need for this population. FWD1802 is a third-generation oral SERD for ER-positive breast cancer patients. It binds competitively to ER, with a much higher affinity than that of fulvestrant. Preclinically, FWD1802 alone and in combination with palbociclib exhibits significant anti-tumor activity in the ER+/HER2- models and in ESR1-mutated models in vitro and in vivo. This ongoing phase I study is to assess the safety, tolerability, pharmacokinetics (PK) and clinical activity of FWD1802 as monotherapy and in combination with palbociclib in ER+/HER2- unresectable locally advanced or metastatic breast cancer with or without ESR1 mutations. Methods: This clinical study (CTR20232130) consists of three parts: monotherapy dose escalation (Part A), dose escalation in combination with palbociclib (Part B) and monotherapy dose expansion in ESR1-mutated patients (Part C). A conventional “3+3” design is used for this study. Approximately a total of 129 patients will be enrolled. FWD1802 is administered orally daily. In monotherapy dose escalation study (Part A), at each dose level, a single ascending dose (SAD) is followed by a 28-day continuous multiple ascending dose (MAD) period (defined as 1 cycle). In Part B dose, palbociclib (125 mg daily) is administered on a 28-day treatment cycle with 21-day on treatment and 7-day off treatment, along with the 28-day treatment cycle of FWD1802. The primary objectives include dose limiting toxicity (DLT), maximum tolerated dose (MTD), safety, tolerability, and the recommended phase 2 dose (RP2D) of FWD1802. Secondary objectives include pharmacokinetics characteristics of FWD1802 and its major metabolite as monotherapy and in combination with palbociclib. Preliminary anti-tumor activity including objective response rate (ORR), duration of response (DoR), disease control rate (DCR) and progression-free survival (PFS) will also be evaluated. DLT evaluation will be conducted after the first cycle of multi-dose period, and tumor assessment will be conducted every 8 weeks by investigators according to RECIST version 1.1. Patients with ER+/HER2- unresectable locally advanced or metastatic breast cancer with or without ESR1 mutation are eligible for the enrollment. Enrolled patients should have progressed disease, or failed from previous therapy, or intolerable or inaccessible to standard therapy. In addition, patients with ECOG score ≤1 and with adequate renal, cardiac, hepatic, and hematologic function are eligible for enrollment. The monotherapy dose escalation (Part A) is currently ongoing. The dose selection for Part B and Part C will be based on the data from the ongoing Part A. Clinical trial information: CTR20232130. Research Sponsor: Shenzhen Forward Pharmaceuticals Co., Ltd. (FORWARD PHARMA). Citation Format: Ling Zeng, Jin Liu, Dan Wu, Huiwen Ge, Zirui Bian, Chenggang Zhu. A phase I open-label dose escalation trial of FWD1802 as monotherapy and in combination with palbociclib in patients with ER+/HER2- unresectable locally advanced or metastatic breast cancer with or without ESR1 mutations [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 PO4-27-05.

Abstract PO2-27-01: TFX06, a next-generation oral CERAN/SERD, shows favorable safety and PK profile, extracranial and intracranial efficacy in patients with ER+/HER2- breast cancer: Preliminary results from a phase 1/2 first-in-human trial

Abstract Aims: TFX06 is an investigational oral complete estrogen receptor (ER) antagonist (CERAN) and selective ER degrader (SERD) (Wu J, et al., 2023 AACR annual meeting). A Phase 1/2, multicenter, open-label dose-escalation and dose-expansion study (CTR20230789) was initiated in China to evaluate TFX06 in previously heavily treated patients with ER+/HER2- locally advanced and/or metastatic breast cancer. The primary objectives were to assess safety and tolerability, recommended Phase 2 dose (RP2D), and pharmacokinetics (PK). Methods: A 3+3 dose-escalation design was implemented. TFX06 tablets were orally administered at 60 mg/100 mg daily for 28 days of treatment cycle until disease progression or intolerant to TFX06. As of the cut-off date (Sept. 15, 2023), the Phase 1 study enrolled five patients with Stage IV diseases with distant metastases. Three were treated with TFX06 at 60 mg, the starting dose and two at the next dose level of 100 mg. Median age of patients were 50 years (39-65) with a ECOG score of 0-1. Two out of five patients carried estrogen receptor 1 (ESR1) mutations (ctDNA). Prior anticancer treatments included one or two lines of endocrine therapies, including fulvestrant, and/or one or two lines of chemotherapies. Clinical responses by investigator according to RECIST v1.1 were performed every two cycles to explore preliminary efficacy. Results: Three patients in 60 mg and one in 100 mg dose cohorts completed dose-limiting toxicity (DLT) observation period. No DLT observed. All treatment related adverse events (TRAEs) were grade 1-2 in severity, including nausea, drowsiness, decreases in WBC and neutrophils, and anemia. Analyses of plasma concentrations of TFX06 in all three patients receiving 60 mg revealed a similar PK profile with small variations in Cmax. The average steady-state Cmax and Cmin of TFX06 were 101 ng/mL and 65 ng/mL, respectively, and the Cmax of TFX06 was approximately 4-fold higher than that of fulvestrant administered intramuscularly at 500 mg in clinic (28 ng/mL). Two patients in the 60 mg cohort were evaluable for response assessment. One patient discontinued due to progressive disease (PD) after two cycles of treatment. This patient experienced a 17.1% decrease in target lesions in the lung; however, new metastases to bone observed. The other patient who carried ESR1 D538G mutation showed a decrease of 91% in ESR1 mutation burden 8 days after treatment, and a decrease of 34.2% in target lesions (partial response, PR), including a substantial shrinkage of 78.6% in the brain lesion (target lesion), as well as complete responses (CR) of non-target lesions in lung after 4 cycles of treatment. This patient remains on the study as of the cut-off date. Conclusions: Based on the preliminary results, oral administration of TFX06 at 60 or 100 mg once daily had favorable safety and PK profiles. TRAEs, such as gastrointestinal toxicities, bradycardia, and visual disturbances were not observed. Pleasingly, TFX06 demonstrated early extracranial and intracranial antitumor activity (PR) in a patient with a brain metastasis. Citation Format: Zhaojian Fu, Zhiye Zhang, Jia Song, Yang Chen, Douglas Fang, Wei Sha, Jian Zhang, Charles Ding. TFX06, a next-generation oral CERAN/SERD, shows favorable safety and PK profile, extracranial and intracranial efficacy in patients with ER+/HER2- breast cancer: Preliminary results from a phase 1/2 first-in-human 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 PO2-27-01.

Abstract 4626: A kit targeting detection of ESR1 mutations from circulating exosomal RNA and cell-free DNA supports longitudinal studies into endocrine therapy resistance in a broadly accessible RT-qPCR format

Abstract Introduction: The most prevalent subtype of breast cancer is HR+/HER2- (positive for hormone receptor, negative for human epidermal growth factor receptor 2). For metastatic breast cancer (mBC), a frequent occurrence is resistance to the aromatase inhibitors used in endocrine therapy (ET). Resistance is mediated through mutations in the ligand binding domain of estrogen receptor 1 (ESR1). Approval of elacestrant, a second-line agent for HR+/HER2-/ESR1 mutated mBC , underscores the need for early detection of ESR1 resistance mutations. We have developed sensitive RT-qPCR reagents for targeted longitudinal studies into the emergence of ESR1 resistance mutations in plasma. This all-inclusive kit analyzes circulating exosomal RNA (exoRNA) and cell-free DNA (cfDNA) to accurately identify low copy number mutations with a rapid turnaround time. Methods: Challenge panels were constructed using blends of synthetic nucleic acids of known mutational status in a background of partially fragmented wild-type DNA from cell lines. Additionally, plasma samples collected from subjects with stage IV mBC (HR+/HER2-) on active aromatase inhibitor therapy, +/- CDK 4/6 inhibitor for a minimum of one year, were subjected to an in-house method optimized to co-enrich exoRNA and cfDNA. Multiplex RT-qPCR-based target enrichment performed on 11 ESR1 mutations and an internal control were evaluated on widely used thermal cycler and qPCR instruments. Results: We developed technology that interrogates 11 key ESR1 mutations associated with ET resistance in HR+/HER2- mBC cases. Earlier studies performed in simplex analyzed plasmid-based ESR1 mutations with detection of 10 or fewer mutant copies, estimating analytical sensitivity of at least 0.1% (5 mutant copies in a background of 5,000 WT copies). Herein, we describe the subsequent work to complete the methodology employed in the kit, including the incorporation of an endogenous control, external batch run control materials (positive, negative), deeply optimized reagents (RT, pre-amplification, qPCR), and automated analysis. We report on the results of studies into the kit’s performance characteristics against their targets: analytical sensitivity of the equivalent of 5 copies of mutation per mL plasma, high precision across replicates at 0.1% allele fraction, analytical specificity of ≥90% negative results in mutation-negative replicates, as well as workflow attributes (reduced pipetting steps and turnaround time). Conclusion: Our solution to detect ESR1 resistance mutations facilitates the generation of nucleic acid sample to results within one day using liquid biopsy samples and a multiplexed RT-qPCR. Enhancing mutation detection sensitivity by complementing cfDNA with exoRNA will facilitate future research into breast cancer treatment options. Citation Format: Justin T. Brown, Julie R. Thibert, Liangjing Chen, Melissa Church, Holli Dale, Jamie Myers, Elliot Hallmark, Megan Yociss, J Aquiles Sanchez, Kurt Franzen, Johan Skog, Gary Latham, Brian Haynes, Sarah Statt. A kit targeting detection of ESR1 mutations from circulating exosomal RNA and cell-free DNA supports longitudinal studies into endocrine therapy resistance in a broadly accessible RT-qPCR format [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4626.

Cost-effectiveness analysis of elacestrant versus standard endocrine therapy for second-/third-line treatment of patients with HR+/HER2- advanced or metastatic breast cancer: a US payer perspective.

This study evaluated the cost-effectiveness of elacestrant (ELA) and standard-of-care (SOC) as second-/third-line treatment for pretreated estrogen receptor (ER)- positive/human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer (A/MBC) in the US. The 3 health states partitioned survival model (PSM) was conducted from the perspective of the US third-party payers. The time horizon for the model lasted 10 years. Effectiveness and safety data were derived from the EMERALD trial (NCT03778931). Costs were derived from the pricing files of Medicare and Medicaid Services, and utility values were derived from published studies. One-way sensitivity analysis as well as probabilistic sensitivity analysis were performed to observe model stability. ELA led to an incremental cost-effectiveness ratio (ICER) of $8,672,360/quality-adjusted life year (QALY) gained compared with SOC in the overall population and $2,900,560/QALY gained compared with fulvestrant (FUL) in the ESR1(estrogen receptor 1) mutation subgroup. The two ICERs of ELA were significantly higher than the willingness-to-pay (WTP) threshold values of $150,000/QALY. ELA was not cost-effective for the second-/third-line treatment of patients with ER+/HER2-A/MBC compared with SOC in the US.

ESR1 mutational landscape and impact of co-existing resistance variants on clinical outcomes in patients with metastatic breast cancer.

1074 Background: Mutations in estrogen receptor 1 ( ESR1) confer resistance to aromatase inhibitors but may retain sensitivity to selective estrogen receptor degraders (SERD). Recently, elacestrant, an oral SERD, was approved for patients with HR+/HER2- ESR1mut metastatic breast cancer (MBC). In this study, we evaluated the genomic landscape of ESR1 alterations from a genomic database and also evaluated the impact of resistance-associated alterations on clinical outcomes. Methods: A large de-identified database of mutations detected in plasma cell-free DNA (cfDNA) from patients with HR+/HER2- MBC was assessed for ESR1 mutations and resistance-associated co-alterations. We used the Guardant 360 assay, a 70- to 74-gene targeted next generation sequencing panel and applied a breast cancer subtype classifier based on detected mutations (Bardia A, SABCS 2020). In addition, clinical outcomes were evaluated from a clinically annotated institutional dataset, linking genomic alterations with progression-free survival (PFS) and overall survival (OS) to single-agent SERD therapy for patients with MBC. Results: Among 11,456 patients with MBC and detectable cfDNA, 4,694 were classified as likely HR+/HER2-, of which 2,708 (58%) had ESR1 missense mutations, the majority of which occurred in the ligand binding domain (n = 2,690, 99%). Among the latter, alterations included 204 unique mutations, of which 55 were variants of uncertain significance (VUS). Multiple ESR1 mutations were detected in 979 (36%) patients. Among 567 patients classified as HR+/HER2- that underwent serial sampling, 136 (24%) had detectable ESR1 missense mutations that were previously undetectable. Within the ESR1mut population, 1,990 (74%) had co-existing non-synonymous, non-VUS alterations at genes associated with SERD resistance: PIK3CA (n = 1,330; 49%), ARID1A (n = 314; 12%), PTEN (n = 201; 7%), ERBB2 (n = 180; 7%), AKT1 (n = 139; 5%), and NF1 (n = 103; 4%); top amplifications were FGFR1 (n = 522; 19%), EGFR (n = 277; 10%), and MYC (n = 177; 7%). Among a clinically annotated HR+/HER2- subgroup (n = 350), 51 patients received SERD monotherapy, either fulvestrant (n = 9) or oral SERD on clinical trial (n = 42); among 28 patients with cfDNA variants associated with SERD resistance, median PFS and OS were lower compared to the 23 patients without resistance mutations (PFS: 2.4 vs 11.3 mo; HR = 0.44; 95% CI, 0.23-0.81; p = 0.008; and OS: 21.1 vs 31.4 mo; HR 0.47; 95% CI, 0.22-0.98; p = 0.045). Conclusions: ESR1 mutations that qualify patients to receive approved SERD therapy are detectable by plasma-based genotyping and can associate with various genomic co-alterations, including variants that may impact clinical outcomes. Further research is needed to confirm the impact of co-alterations in additional datasets and evaluate the role of SERD-based combination therapy to further improve clinical outcomes of patients with MBC.

Abstract P2-03-22: Assessment of the relationship between duration of treatment with hormonal therapy and the detection of ESR1 mutations in hormone-receptor positive, HER2 negative, breast cancer

Abstract Background Hormonal therapeutic agents such as the selective estrogen receptor modulator, tamoxifen, as well as the aromatase inhibitors (AIs), anastrozole, exemestane, and letrozole, are a mainstay in the treatment of patients with hormone-receptor positive (HR+) breast cancer. Despite the effectiveness of hormonal therapy in the treatment of patients with HR+ breast cancer, acquired resistance to both AIs and tamoxifen does occur, often associated with mutations of the estrogen receptor 1 (ESR1). Although the acquisition of ESR1 mutations in patients with HR+ breast cancer treated with hormonal therapy has been established as a mechanism of resistance, there is a paucity of data on the relationship between the duration of treatment with hormonal therapies and the development of ESR1 mutations. Using a cohort of patients with HR+ breast cancer who underwent next generation sequencing (NGS) as part of routine clinical care, we aim to assess the relationship between the duration of exposure to aromatase inhibitors and the detection of ESR1 mutations. The results of this study will further our understanding of ESR1-mutations and provide insight into strategies to optimize therapy for patients with HR+ breast cancers. Methods This is a retrospective analysis of patients 18 years of age or older treated at a single academic cancer center in the USA, diagnosed with HR+, HER2-negative breast cancer and who have received somatic NGS as part of clinical care over a six-year period through July 2019. Demographic, disease-related, and treatment related variables were collected along with genomic testing variables (ie. ESR1-mutation status) for each patient who met the inclusion criteria. Two patient groups were formed on the basis of ESR1-mutation status (present vs. absent) and compared on the basis of duration of treatment with AI, and other clinical and demographic categories. Results A total of 247 patients who were diagnosed with HR-positive, HER2-negative breast cancer and received NGS were identified and are included in this analysis. A total of 138 (56%) patients were assessed via tissue based NGS and 109 (44%) by liquid biopsy. The median age at time of sequencing was 58 years [57 years (ESR1-absent) vs. 60 years (ESR1-present); p =0.009]. The majority of patients were white (83%) and post-menopausal (57%). Invasive ductal carcinoma was the most common histological type (82%). Nearly all (96%) patients had stage IV disease at time of sequencing and ESR1 mutations were found exclusively in those with stage IV disease. ESR1 mutations were identified in 62 patients (25%). The median duration of treatment with aromatase inhibitors prior to testing was 19.3 months in both groups (ESR1 mutations absent vs. detected). There was no difference in prior tamoxifen use between groups (absent 43%, present 48%; p=0.48). In patients tested by tissue based NGS, ESR1 mutations were more frequently detected in metastatic sites (28% vs. 13%; p=0.03). Twenty-two patients for whom NGS did not identify an ESR1 mutation prior to July 2019 received subsequent NGS testing. Of these 22 patients, six (27%) had ESR1 mutations detected by subsequent NGS. In these six patients, ESR1-mutations were detected by NGS a median of 2.2 years (range 1.7 years to 2.6 years) after prior NGS. Conclusion There was no difference in duration of treatment with aromatase inhibitor therapy in patients with ESR1 mutations vs. those without ESR1 mutations detected by NGS. Patients with ESR1 mutations tended to be older. Although the detection of ESR1 mutations is associated with acquired therapy resistance and progression on treatment, a longer duration of treatment may make the emergence of ESR1 mutations more likely. These counterbalancing factors may have contributed to the results reported here. Citation Format: Hannah Ayettey-Anie, Kamran A. Ahmed, Iman Washington, Kosj Yamoah, Todd Knepper. Assessment of the relationship between duration of treatment with hormonal therapy and the detection of ESR1 mutations in hormone-receptor positive, 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 P2-03-22.

Abstract P5-06-04: The prognostic value of c-MET monitoring by using c-MET-enriched circulating tumor cells in HR-positive HER2-negative metastatic breast cancer

Abstract [Background] As the development of endocrine resistance and late recurrences are the major clinical concerns in hormone receptor (HR)-positive/HER2-negative metastatic breast cancer (MBC) patients, biomarkers to predict the occurrence of endocrine resistance or disease progression are crucial for improving patient outcomes. Aberrant HGF/c-MET signaling pathway has been reported to play a role in various cellular processes in cancer. Estrogen Receptor 1 (ESR1) mutations, encoding estrogen receptor α, are associated with endocrine resistance in HR+ breast cancer. PIK3CA hotspot mutations that induce hyperactivation of the PI3K are found in 30-40% of HR+ advanced breast cancers. In this context, we evaluated the prognostic values of c-MET-enriched CTC, ESR1 mutations, PIK3CA mutations, and cfDNA concentrations detected in the blood of HR+HER2- MBC patients. [Methods] MBC patients were prospectively enrolled during standard treatments at Samsung Medical Center (IRB No.2019-08-119). Circulating tumor cells (CTCs) were isolated using the GenoCTC® with c-MET-enriched or EpCAM-enriched CTC isolation kits (Genobio Corp., South Korea) from 4mL of blood each. PIK3CA and ESR1 hotspot mutations were analyzed by droplet digital PCR kits (Gencurix Inc., South Korea). cfDNA concentrations were calculated using ESR1 gene copy numbers from plasma. To compare the proportion of c-MET overexpression between primary breast tumors and metastatic sites in HR+HER2- breast cancer patients, primary breast (n=358) and metastatic sites (n=27) were independently collected. c-MET expression was evaluated by an immunohistochemistry assay using an anti-total c-MET (SP44) antibody with a Ventana Discovery XY automated system according to the manufacturer’s instruction. c-MET overexpression was defined if the staining was scored as 2+ or 3+. Progression-free survival (PFS) was defined as the time from blood draw to the first of either disease progression or death during standard therapy. [Results] Out of 93 patients with HR+ MBC, analysis was performed in 63 HR+HER2- MBC patients. Seventeen patients (27%) had one or more EpCAM-enriched CTCs, and fourteen patients (22%) had one or more c-MET-enriched CTCs detected in their blood. The median follow-up time and median time to censoring were 8.4 months and 18.7 months, respectively. According to the Kaplan-Meier survival analysis by log-rank test, c-MET-enriched CTCs, cfDNA concentrations, and ESR1 mutations were significantly associated with PFS (p=0.0026, 0.0064, and 0.011, respectively). However, PIK3CA mutations and EpCAM-enriched CTCs were not statistically significant with PFS (p=0.38 and 0.86, respectively). Multivariate analysis showed that both c-MET-enriched CTCs (HR=3.5, p=0.014) and cfDNA concentrations (HR=2.2, p=0.031) were independent predictors for PFS in HR+HER2- MBC. The proportion of c-MET overexpression was significantly higher in metastatic sites (22.2%) than in primary breast tumors (4.7%) in HR+HER2- breast cancer patients (p=0.00002). As c-MET-enriched CTCs and cfDNA concentrations were independent predictors of disease progression, patients were divided into two groups depending on the result of c-MET-enriched CTCs and cfDNA concentration. When patients with low c-MET-enriched CTC and cfDNA concentrations were classified as a low-risk group and other patients into a high-risk group, the high-risk group had a shorter PFS than the low-risk group (p=0.003). [Conclusion] This study provided c-MET-enriched CTCs and cfDNA concentrations calculated by ESR1 copy numbers in patient blood were significant independent predictors of disease progression in HR+HER2- MBC. The poor prognosis in the c-MET-enriched CTC-high group and the difference in the c-MET overexpression rate between the primary breast and metastatic sites suggested the importance of monitoring c-MET-enriched CTCs in the blood of HR+HER2- MBC patients. Citation Format: Jieun Park, Eun Sol Chang, Ji-Yeon Kim, Chaithanya Chelakkot, Minjung Sung, Ji-Young Song, Kyungsoo Jung, Na Young Kim, Hyegyeong Lee, Mi-Ran Kang, Yeon Hee Park, Young Kee Shin, Yoon-La Choi. The prognostic value of c-MET monitoring by using c-MET-enriched circulating tumor cells in HR-positive HER2-negative metastatic 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 P5-06-04.

Abstract P5-05-04: Estrogen receptor 1 (ESR1) mutations in circulating tumor DNA (ctDNA) from patients with ER+/HER2- metastatic breast cancer (mBC) treated with lasofoxifene or fulvestrant in the ELAINE 1 study

Abstract Introduction: Acquired ESR1 mutations (mutESR1) after long-term endocrine therapy drive treatment resistance, metastasis, and poor prognosis for patients (pts) with ER+/HER2- metastatic breast cancer (mBC). Lasofoxifene (LAS), a selective estrogen receptor modulator, alone or with a CDK4/6 inhibitor (CDK4/6i) reduced tumor growth better than fulvestrant (Fulv) in mutESR1 BC xenograft models. ELAINE 1 is a randomized trial of LAS vs Fulv in pts with mutESR1 and prior progression on aromatase inhibitor and CDK4/6i. Preliminary results (ESMO 2022) showed that LAS prolonged median progression-free survival (mPFS) compared with Fulv with a favorable safety profile. Here, we report changes in ESR1 ctDNA mutant allele frequency (MAF) from baseline to 8 wks and their associations with clinical benefit (CB) and mPFS. Methods: ELAINE 1 pts were randomized to oral LAS 5 mg daily or IM Fulv 500 mg on days 1, 15, and 29, then every 4 wks, until disease progression or severe toxicity. ctDNA mutESR1 mutations (baseline and 8 wks) were assessed using the Sysmex Inostics OncoBeam or SafeSeq assays—which detect mutESR1 at low allele fractions. MAF changes from baseline to wk 8 were characterized as decreased (decrease in ESR1 MAF or fully cleared), increased (increase in MAF), or equivocal (polyclonal patients [>1 mutESR1] with some increasing and decreasing MAF trends); correlations with PFS and CB were explored. Efficacy measures included objective response rate (ORR), PFS, and CB at 24 wks (CB defined as response or stable disease ≥24 wks). Results: 103 pts received LAS (n=52) or Fulv (n=51). Most common baseline ESR1 variants detected were D538G (56%), Y537S (39%), Y537N (29%), E380Q (22%); 56 (54%) pts were polyclonal. Of the 61 pts with evaluable baseline and wk 8 ctDNA, LAS decreased mutESR1 MAF in 29/35 pts (83% [11 complete clearance]) while Fulv decreased mutESR1 MAF in 16/26 pts (61.5% [6 complete clearance]) (Table). mPFS with LAS was 8 and 4 mos for pts with decreased/cleared MAF and increased MAF, respectively, and with Fulv was 4.5 and 2.8 mos, respectively (Table). LAS decreased the common mutESR1 variants more frequently than Fulv (median relative change -87.1% vs -14.7%). In pts with decreased MAF, CB was observed in 16/29 LAS pts (55%) and 4/16 Fulv pts (25%). The predictiveness of ESR1 MAF clearance for CB was also explored. Of 11 pts with ESR1 MAF clearance taking LAS, 10 achieved CB, yielding a positive predictive value (PPV) of 90.9%. In contrast, 2/6 pts with ESR1 MAF clearance taking Fulv had CB for a PPV of 33.3%. Sensitivity for predicting CB based on direction of ESR1 MAF change was 94% with LAS and 80% with Fulv. In pts with Y537S MAF (n=33), LAS decreased Y537S in 13/15 (87%), with a median relative MAF decrease of 89%. In marked contrast, Fulv increased Y537S MAF in 11/18 pts (61%), corresponding to an MAF relative increase of 82%. LAS and Fulv resulted in complete clearance of Y537S MAF in 33% and 6% of pts, respectively. Conclusion: Our data demonstrate that LAS more effectively decreased or cleared mutESR1 than Fulv. Further, mutESR1 clearance was associated with prolonged PFS and more CB in LAS but not Fulv pts, suggesting that LAS results in robust mutESR1 target engagement. Taken together, our data suggest mutESR1 as a potential liquid biomarker for predicting response to LAS in mutESR1, endocrine-resistant mBC pts. Table. Change from baseline to week 8 in ESR1 MAF and clinical benefit at 24 weeks. CI, confidence interval; MAF, mutant allele fraction; ND, none detected; PFS, progression-free survival. Citation Format: Matthew P. Goetz, Einav Gal-Yam, Daniel Stover, Sarah L. Sammons, Stephanie L. Graff, Grace Wang, Massimo Cristofanilli, Gary Riordan, Hillary S. Sloane, Dominic Carroll, Paul V. Plourde, David J Portman. Estrogen receptor 1 (ESR1) mutations in circulating tumor DNA (ctDNA) from patients with ER+/HER2- metastatic breast cancer (mBC) treated with lasofoxifene or fulvestrant in the ELAINE 1 study [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 P5-05-04.

Abstract P5-05-02: Estrogen receptor 1 (ESR1) mutations in circulating tumor DNA (ctDNA) from patients with ER+/HER2- metastatic breast cancer (mBC) treated with lasofoxifene plus abemaciclib in the ELAINE 2 study

Abstract Introduction: Use of long-term endocrine therapy (ET) for ER+ breast cancer often leads to acquired ESR1 mutations (mutESR1), causing endocrine resistance, tumor progression, and poor prognosis. An unmet clinical need exists for treating ER+ mBC patients with mutESR1, particularly after progression on CDK4/6 inhibitors (CDK4/6i). ELAINE 2 is an open-label, phase 2, multicenter trial evaluating safety and efficacy of lasofoxifene (LAS [selective estrogen receptor modulator]) plus abemaciclib (Abema [CDK4/6i], provided by Eli Lilly) in patients with ER+/HER2- and mutESR1 mBC who progressed after prior ET. Preliminary data with LAS plus Abema showed median progression-free survival of 55.7 wks, objective response rate of 50%, and 24-wk clinical benefit (CB) rate of 69%, with an acceptable safety and tolerability profile. Here, we report ESR1 ctDNA mutant allele frequency (MAF) and correlations of ESR1 MAF changes with CB. Methods: ELAINE 2 patients with detectable ctDNA mutESR1 at baseline (BL) were analyzed. Oral LAS 5 mg/day and Abema 150 mg BID were taken until disease progression, death, unacceptable toxicity, or withdrawal from the study. ctDNA was assessed by the Sysmex-Inostics SafeSeq assay—which detects mutESR1 at low allele fractions—at BL, every 4 wks, and end of treatment. MAF changes from BL to wk 4 were characterized as decreased (decrease in ESR1 MAF or none detected [ND]), increased (increase in MAF), or equivocal (in polyclonal patients [>1 mutESR1] with some MAF increasing and decreasing trends). Correlations of MAF change at 4 wks with CB at 24 wks were explored. Results: 29 patients (median of 2 prior metastatic therapies: 97% CDK4/6i, 79% fulvestrant, 48% chemotherapy) had BL mutESR1 of Y537S (66%), D538G (45%), Y537N (28%), and other less frequently detected mutations; 14 (48.3%) patients were polyclonal. 26 of 29 patients had evaluable BL and wk-4 ctDNA results: 21 patients had decreased MAF (81% [54% with ND]), 3 (12%) had increased, and 2 (8%) had equivocal ESR1 MAF changes (Table). CB at 24 wks was seen in 17 patients with a decrease, 2 with an increase, and 1 with equivocal MAF change. A sensitivity of 89.5% and specificity of 20% were calculated for predicting CB based on direction of ESR1 MAF change. The positive predictive value (PPV) for CB with decreased MAF was 81% and the negative predictive value (NPV) for an increased MAF was 33%. Of the 14 (54%) patients with ND ESR1 MAF, 13 had CB resulting in 87% sensitivity, 50% specificity, 93% PPV, and 33% NPV. Conclusion: In ELAINE 2, 81% of patients had decrease/cleared (ND) mutESR1 after 4 wks of LAS plus Abema, which correlated with clinical benefit. All mutESR1 detected appear targeted with this therapy. High sensitivity and favorable PPV were observed in patients with decreased MAF, and even more so in those with ND MAF; however, increased MAF was less specific and not as predictive of treatment failure. Our results indicate that ESR1 liquid biopsy evaluation may be an adequate non-invasive surrogate marker for monitoring patients on treatment. Further study in a larger population of women with endocrine-resistant mBC and acquired mutESR1 is warranted to explore this potential for monitoring treatment response or resistance to this novel LAS-Abema combination. Table. Change from baseline to week 4 in ESR1 MAF and clinical benefit at 24 weeks. CI, confidence interval; MAF, mutant allele fraction; ND, none detected; NPV, negative predictive value; PPV, positive predictive value. *Sensitivity and specificity analyses do not include equivocal results. Citation Format: Senthil Damodaran, Halle Moore, Ciara C. O’Sullivan, Paul V. Plourde, Gary Riordan, Hillary S. Sloane, Debu Tripathy, Dominic Carroll, David J Portman. Estrogen receptor 1 (ESR1) mutations in circulating tumor DNA (ctDNA) from patients with ER+/HER2- metastatic breast cancer (mBC) treated with lasofoxifene plus abemaciclib in the ELAINE 2 study [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 P5-05-02.

LBA20 Open-label, randomized study of lasofoxifene (LAS) vs fulvestrant (Fulv) for women with locally advanced/metastatic ER+/HER2- breast cancer (mBC), an estrogen receptor 1 (ESR1) mutation, and disease progression on aromatase (AI) and cyclin-dependent kinase 4/6 (CDK4/6i) inhibitors

Acquired ESR1 mutations cause endocrine resistance, driving metastasis and poor prognosis in patients with ER+/HER2- mBC. A phase 2 trial of LAS plus abemaciclib (ELAINE 2) showed efficacy in heavily pretreated patients with ESR1-mutated mBC post-CDK4/6i (ASCO 2022). Here we describe ELAINE 1, a randomized trial of LAS vs Fulv in a post-CDK4/6i second-line setting. Women with ER+/HER2-/ESR1-mutated mBC and progression on prior (≥12 mos) AI plus CDK4/6i (n=103) were randomized to oral LAS 5 mg (n=52) daily or IM Fulv 500 mg (n=51) days 1, 15, and 29, then every 4 weeks, until disease progression or severe toxicity. Imaging occurred every 2 mos (or if clinically indicated). Primary endpoint was progression-free survival (PFS). Mean age was 60.8 yr (33-84); 83% were white, 66% had visceral disease, 71% (n=73) had measurable disease. For LAS vs Fulv, median PFS was 6.04 mos (95% CI, 2.82–8.04) vs 4.04 mos (95% CI, 2.93–6.04), P=0.138 (HR, 0.699 [95% CI, 0.445–1.125]); PFS at 12 mos was 30.7% vs 14.1%; clinical benefit rate was 36.5% vs 21.6%, P=0.12. Objective response rate for LAS vs Fulv was 13.2% vs 2.9%, P=0.12, with 1 complete response (60-week duration) and 4 partial responses (PR) in the LAS arm versus 1 PR in the Fulv arm. PFS was numerically and consistently greater with LAS vs Fulv when visceral metastasis and/or Y537S ESR1 mutation subgroups were analyzed. Clearance of ctDNA also favored LAS over Fulv. Most common adverse events were fatigue, nausea, arthralgias, and hot flushes; most were Grade 1/2. No thrombotic events occurred. ELAINE 1 is the first clinical trial comparing LAS with Fulv in ESR1-mutated mBC patients with progression on CDK4/6i and demonstrating activity of a novel SERM in this setting. All clinical outcomes numerically favored LAS vs Fulv in this signal-seeking study. LAS may be a new treatment option following endocrine/CDK4/6i therapies if efficacy is confirmed in a larger, adequately powered clinical study. A phase 3 combination study of LAS and abemaciclib is planned based on encouraging efficacy/safety in ELAINE 2.

Open-label, phase 2, multicenter study of lasofoxifene (LAS) combined with abemaciclib (Abema) for treating pre- and postmenopausal women with locally advanced or metastatic ER+/HER2− breast cancer and an ESR1 mutation after progression on prior therapies.

1022 Background: Resistance to endocrine therapy can develop when treating estrogen receptor positive (ER+), metastatic breast cancer (mBC). Treatment with aromatase inhibitors can lead to acquired mutations in the estrogen receptor 1 ( ESR1), which can constitutively activate the ER, leading to endocrine resistance and worse disease prognosis. Treatment options for mBC patients with an ESR1 mutation are limited. Further, data suggest that patients could derive clinical benefit from Abema after progression on prior cyclin-dependent kinase 4/6 inhibitor (CDK4/6i). LAS, a third-generation selective estrogen receptor modulator, as monotherapy or combined with a CDK4/6i, was shown to have superior efficacy over fulvestrant (FVT) in preclinical breast cancer models expressing ESR1 mutations. Based on these results, a phase 2 clinical trial of LAS combined with Abema was initiated in mBC patients with ESR1 mutations. Methods: ELAINE 2 is an open-label, phase 2, multicenter trial evaluating the safety and efficacy of LAS combined with the CDK4/6i, Abema. Study participants were pre- and postmenopausal women with ER+/HER2- mBC with acquired ESR1 mutation (identified by ctDNA testing), whose disease had progressed on one or two lines of hormonal therapy for metastatic disease with or without a CDK4/6i (including Abema). Patients took oral LAS 5 mg/day and Abema 150 mg BID. Treatment continued until evidence of disease progression, death, unacceptable toxicity, or withdrawal from the study. The primary endpoint was safety, and secondary endpoints were progression free survival (PFS), objective response rate (ORR), and clinical benefit rate (CBR). Results: 29 patients were enrolled at 16 US sites (Oct 2020 to June 2021). Mean age was 58.3 y (35-79 y); 86% were Caucasian. Most had progressed with at least 2 previous hormonal treatments (80%). All except 1 patient received a prior CDK4/6i and 72% had received prior FVT; 48% had chemotherapy in the metastatic setting. Four patients discontinued the trial due to adverse events (AEs, n = 2), consent withdrawal (n = 1), or investigator withdrawal (n = 1). No deaths occurred during the study and few Grade 3/4 AEs were observed. Most common AEs were diarrhea, nausea, and leukopenia. Five patients had an Abema dose reduction from 150 mg to 100 mg BID. To date, 11 patients have progressed and 14 continue treatment. The censored median PFS was 13.9 mos (95% CI, 8.0‒NE), the ORR 33.3% (95% CI, 16.3‒56.3) with 6 confirmed partial responses, and the CBR 62.1% (95% CI, 44.0‒77.3). Conclusions: LAS combined with Abema in the ELAINE 2 trial was well tolerated and demonstrated robust and meaningful efficacy in women with ER+/HER2- mBC and an ESR1 mutation who had progressed on previous CDK4/6i therapies. Clinical trial information: NCT04432454.

Abstract OT2-16-01: The SMILE study: A phase 2 trial of onapristone in combination with fulvestrant for patients with ER+ and HER2- metastatic breast cancer after progression on endocrine therapy and CDK4/6 inhibitors

Abstract Background Antiprogestins, including selective progesterone receptor (PgR) modulators (SPRMs) that act as PgR antagonists, are a promising class of therapeutics for overcoming endocrine resistance including in patients who develop activating estrogen receptor 1 (ESR1) mutations after prior endocrine therapy (ET). The SMILE study (NCT04738292) is a multi-institutional phase II clinical trial to determine the efficacy and safety of an antiprogestin, onapristone in combination with fulvestrant as second-line therapy for patients with ER+,PgR+, HER2 -metastatic breast cancer (MBC). Methods Patients with locally advanced or MBC who progress on ≥2 lines of prior therapy with ET as a single agent or in combination with CDK4/6 inhibitors, mTOR inhibitors and one line of chemotherapy in the metastatic setting are eligible. Other criteria include ECOG performance status ≤ 2, measurable disease per RECIST 1.1 criteria, adequate organ function, availability of an archived tumor biopsy block confirming the diagnosis of MBC, optional biopsy at disease progression on trial, and optional 18F-fluorofuranylnorprogesterone (18F-FFNP) PET/CT imaging. Ovarian suppression is allowed. Prior ET with tamoxifen or aromatase inhibitor therapy in the adjuvant or metastatic setting is allowed. Patients with prior exposure to fulvestrant, antiprogestins, or CDK inhibitors in the adjuvant setting are ineligible. Following consent, all subjects will receive fulvestrant at the recommended dosing schedule plus onapristone 50 mg orally, twice daily, until disease progression, unacceptable toxicity, or discontinuation for any other reason. Using the Simon Two-Stage design, a total of 39 patients will be enrolled over two years from 6-8 sites within Wisconsin Oncology Network (WON). In the first stage of the trial, 21 subjects will be enrolled; if ≥2 responses are observed, then 18 additional subjects will be enrolled in the second stage. This design yields a one-sided type I error rate of 5% if the ORR is 7% and power of 80% when the combination's true response rate is 20%. The primary objective is to evaluate the objective response rate; secondary objectives include safety and tolerability, progression-free survival, disease control rate, and duration of response. Other correlates include optional functional imaging of PgR binding with 18F-FFNP PET/CT and biomarker analysis (ER, total PgR, HER2, Ki67, CD24, CD44, LDH1, KLF4, CK 5/6, PhosphoSer294-PgR on tissue samples, ESR1 mutations, and circulating tumor DNA analysis). The study was approved in January 2021, and enrollment is active at the time of this submission. Citation Format: Sailaja Kamaraju, Amy Fowler, Chung Yee Cheng, Lubna N Chaudhary, John Burfeind, Janet Retseck, Amye J Tevaarwerk, Mark Burkard, Elisavet Paplomata, Amanda M Parkes, Julie M Jorns, Sergey Tarima, Douglas Yee, Hallgeir Rui, Carol Lange, Tarek Sahmoud, Kari B Wisinski. The SMILE study: A phase 2 trial of onapristone in combination with fulvestrant for patients with ER+ and HER2- metastatic breast cancer after progression on endocrine therapy and CDK4/6 inhibitors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-16-01.

Next-generation selective estrogen receptor degraders and other novel endocrine therapies for management of metastatic hormone receptor-positive breast cancer: current and emerging role.

Endocrine therapy (ET) is a pivotal strategy to manage early- and advanced-stage estrogen receptor-positive (ER+) breast cancer. In patients with metastatic breast cancer (MBC), progression of disease inevitably occurs due to the presence of acquired or intrinsic resistance mechanisms. ET resistance can be driven by ligand-independent, ER-mediated signaling that promotes tumor proliferation in the absence of hormone, or ER-independent oncogenic signaling that circumvents endocrine regulated transcription pathways. Estrogen receptor 1 (ESR1) mutations induce constitutive ER activity and upregulate ER-dependent gene transcription, provoking resistance to estrogen deprivation and aromatase inhibitor therapy. The role ESR1 mutations play in regulating response to other therapies, such as the selective estrogen receptor degrader (SERD) fulvestrant and the available CDK4/6 inhibitors, is less clear. Novel oral SERDs and other next-generation ETs are in clinical development for ER+ breast cancer as single agents and in combination with established targeted therapies. Recent results from the phase III EMERALD trial demonstrated improved outcomes with the oral SERD elacestrant compared to standard anti-estrogen therapies in ER+ MBC after prior progression on ET, and other agents have shown promise in both the laboratory and early-phase clinical trials. In this review, we will discuss the emerging data related to oral SERDs and other novel ET in managing ER+ breast cancer. As clinical data continue to mature on these next-generation ETs, important questions will emerge related to the optimal sequence of therapeutic options and the genomic and molecular landscape of resistance to these agents.

ESR1 Gene Mutation in Hormone Receptor-Positive HER2-Negative Metastatic Breast Cancer Patients: Concordance Between Tumor Tissue and Circulating Tumor DNA Analysis.

Endocrine therapy represents the cornerstone of treatment in hormone receptor-positive (HR+), HER2-negative metastatic breast cancer (mBC). The natural course of this disease is marked by endocrine resistance, mainly due to Estrogen Receptor 1 (ESR1) acquired mutations. The aim of this study is to evaluate the concordance between ESR1 status in metastatic tumor specimens and matched circulating tumor DNA (ctDNA). Forty-three patients with HR+, HER2-negative mBC underwent both a metastatic tumor biopsy and a liquid biopsy at the time of disease progression. DNA extracted from formalin fixed paraffin embedded (FFPE) tumor specimens and ctDNA from matched plasma were analyzed by droplet digital (dd)PCR for the main ESR1 mutations (Y537S, Y537C, Y537N, D538G, E380Q). We observed a total mutation rate of 21%. We found six mutations on tissue biopsy: Y537S (1), D538G (2), Y537N (1), E380Q (2). Three patients with no mutations in tumor tissue had mutations detected in ctDNA. The total concordance rate between ESR1 status on tumor tissue and plasma was 91%. Our results confirm the potential role of liquid biopsy as a non-invasive alternative to tissue biopsy for ESR1 mutation assessment in mBC patients.

Sensitive quantitation of ESR1 mutations in cell-free DNA from breast cancer patients using base-specific invasive reaction assisted qPCR

Acquired estrogen receptor 1 (ESR1) mutation is being promoted as a key mechanism of resistance to endocrine therapies in breast cancers. It is significative to monitor ESR1 mutations in real time, which provide an opportunity to alter therapy as these mutations emerge. Previous assays based on next-generation sequencing (NGS) and digital PCR (dPCR) usually due to high costs and complicated workflows hampered their clinical adoption in general medical institutions. Here, we proposed a new strategy using base-specific invasive reaction assisted qPCR measure for ESR1 mutations in cfDNA. Two pivotal steps involved in this strategy are target-specific signal generation and the quantification without adding any internal reference or making standard calibration curves. The strategy enabled a high specificity of 0.1% (better than traditional NGS-based method) and a minimum sensitivity of 0.1 copies μL−1. As validation, with the strategy, cfDNA from endocrine therapy-resistant breast cancers and untreated ones were successfully analyzed (20% mutation rate (2/10) with mutation abundance of 0.54–1.65% vs. 0% mutation rate (0/5)). By virtue of cost-effective, highly flexible and precise, the strategy could be readily implemented in general laboratory, showing promising application perspectives in analysis of other types of mutations.

Plasma-Based Longitudinal Evaluation of ESR1 Epigenetic Status in Hormone Receptor-Positive HER2-Negative Metastatic Breast Cancer.

BackgroundEndocrine therapy (ET) is the mainstay of treatment for hormone receptor-positive human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer; however, adaptive mechanisms emerge in about 25–30% of cases through alterations in the estrogen receptor ligand-binding domain, with a consequent ligand-independent estrogen receptor activity. Epigenetic-mediated events are less known and potentially involved in alternative mechanisms of resistance. The aim of this study was to test the feasibility of estrogen receptor 1 (ESR1) epigenetic characterization through liquid biopsy and to show its potential longitudinal application for an early ET sensitivity assessment.MethodsA cohort of 49 women with hormone receptor-positive HER2-negative MBC was prospectively enrolled and characterized through circulating tumor DNA using methylation-specific droplet digital PCR (MS-ddPCR) before treatment start (BL) and after 3 months concomitantly with computed tomography (CT) scan restaging (EV1). ESR1 epigenetic status was defined by assessing the methylation of its main promoters (promA and promB). The most established cell-free tumor DNA (ctDNA) factors associated with ET resistance [ESR1 and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutations] were assessed through next-generation sequencing. Associations were tested through Mann–Whitney U test, matched pairs variations through Wilcoxon signed rank test, and survival was analyzed by log-rank test.ResultsThe ET backbone was mainly based on aromatase inhibitors (AIs) (70.83%) in association with CDK4/6 inhibitors (93.75%). Significantly lower promA levels at baseline were observed in patients with liver metastases (P = 0.0212) and in patients with ESR1 mutations (P = 0.0091). No significant impact on PFS was observed for promA (P = 0.3777) and promB (P = 0.7455) dichotomized at the median while a ≥2-fold increase in promB or in either promA or promB at EV1 resulted in a significantly worse prognosis (respectively P = 0.0189, P = 0.0294). A significant increase at EV1 was observed for promB among patients with PIK3CA mutation (P = 0.0173). A trend was observed for promB in ESR1 wild-type patients and for promA in the ESR1 mutant subgroup.ConclusionThe study proofed the concept of an epigenetic characterization strategy based on ctDNA and is capable of being integrated in the current clinical workflow to give useful insights on treatment sensitivity.

A single droplet digital PCR for ESR1 activating mutations detection in plasma.

Activating mutations in the estrogen receptor 1 (ESR1) gene confer resistance to aromatase inhibitors (AI), and may be targeted by selective estrogen receptor downregulators. We designed a multiplex droplet digital PCR (ddPCR), which combines a drop-off assay, targeting the clustered hotspot mutations found in exon 8, with an unconventional assay interrogating the E380Q mutation in exon 5. We assessed its sensitivity in vitro using synthetic oligonucleotides, harboring E380Q, L536R, Y537C, Y537N, Y537S, or D538G mutations. Further validation was performed on plasma samples from a prospective study and compared with next generation sequencing (NGS) data. The multiplex ESR1-ddPCR showed a high sensitivity with a limit of detection ranging from 0.07 to 0.19% in mutant allele frequency. The screening of plasma samples from patients with AI-resistant metastatic breast cancer identified ESR1 mutations in 29% of them, all mutations being confirmed by NGS. In addition, this test identifies patients harboring polyclonal alterations. Furthermore, the monitoring of circulating tumor DNA using this technique during treatment follow-up predicts the clinical benefit of palbociclib-fulvestrant. The multiplex ESR1-ddPCR detects, in a single reaction, the most frequent ESR1 activating mutations with good sensitivity. This method allows real-time liquid biopsy for ESR1 mutation monitoring in large cohorts of patients.

Evaluation of sanger sequencing method for ESR1 mutation detection in primary breast tumor

Objective: Exon 8 estrogen receptor 1 (ESR1) mutations in the ligand-binding domain play an important role in mechanisms of hormonal therapy resistance in breast cancer. Identification of ESR1 mutations is very important in determining the appropriate steps of therapy. In this study, we evaluate Sanger sequencing technique to detect the ESR1 mutation. Methods: This retrospective study was conducted using 49 advanced breast cancer patients with estrogen receptor positive. Deoxyribonucleic acid (DNA) material was extracted from primary breast tumor samples. Exon 8 ESR1 gene mutation was analyzed by Sanger sequencing method using BigDye Direct Sequencing Kit (Applied Biosystem) with gBlock synthesis gene fragment (Integrated DNA Technologies) D538G as a positive control. Results: The mean age of patients was 46.14 (±9.6) years, and 61.2% were in stage 4. There are no exon 8 ESR1 gene mutations detected in 49 primary tumor samples, whereas the gBlock-positive control showed base substitution in 1613A>G (D538G) indicating the success of sequencing reaction. Conclusion: Sanger sequencing has failed to detect ESR1 mutation in primary tumor breast samples. Other advanced molecular techniques should be performed for diagnosis of primary breast tumors.

Estrogen-independent molecular actions of mutant estrogen receptor 1 in endometrial cancer.

Estrogen receptor 1 (ESR1) mutations have been identified in hormone therapy–resistant breast cancer and primary endometrial cancer. Analyses in breast cancer suggest that mutant ESR1 exhibits estrogen-independent activity. In endometrial cancer, ESR1 mutations are associated with worse outcomes and less obesity, however, experimental investigation of these mutations has not been performed. Using a unique CRISPR/Cas9 strategy, we introduced the D538G mutation, a common endometrial cancer mutation that alters the ligand binding domain of ESR1, while epitope tagging the endogenous locus. We discovered estrogen-independent mutant ESR1 genomic binding that is significantly altered from wild-type ESR1. The D538G mutation impacted expression, including a large set of nonestrogen-regulated genes, and chromatin accessibility, with most affected loci bound by mutant ESR1. Mutant ESR1 is distinct from constitutive ESR1 activity because mutant-specific changes are not recapitulated with prolonged estrogen exposure. Overall, the D538G mutant ESR1 confers estrogen-independent activity while causing additional regulatory changes in endometrial cancer cells that are distinct from breast cancer cells.

Estrogen receptor 1 mutations in 260 cervical cancer samples from Chinese patients.

Cervical cancer is one of the leading causes of cancer-associated mortality among females; however, the underlying molecular mechanisms of its carcinogenesis remain largely unclear. Previous comprehensive genomic studies have revealed prevalent estrogen receptor 1 (ESR1) mutations in breast cancer, which are rare in certain other types of cancer. To the best of our knowledge, it is unknown whether ESR1 mutations also exist in cervical cancer. Considering the evidence that cervical cancer shares certain genetic aberrations with breast cancer, and that the progression of both breast and cervical cancers can be affected by estrogen, it is possible that cervical cancer may also harbor ESR1 mutations. In the present study, a total of 260 Chinese cervical cancer samples with distinct subtypes were tested for the presence of ESR1 mutations. A total of three heterozygous missense ESR1 mutations, p.K303R (c.908A>G), p.T311M (c.932C>T) and p.Y537C (c.1610A>G), were identified in 3/207 (1.4%) cervical squamous cell carcinoma samples, which were absent in 27 adenosquamous carcinomas and 26 adenocarcinomas samples. Of the three individuals with an ESR1mutation, 1 patient was also diagnosed with ovarian endometriosis and the other 2 patients were diagnosed with a uterine fibroid. A bioinformatics analysis suggested that these ESR1 mutations may be pathogenic by promoting the development of cervical cancer. Furthermore, a previous comprehensive study confirmed that individuals with cervical squamous cell carcinoma possessed ESR1 mutations. These combined studies indicate that ESR1 mutations may participate in the carcinogenesis of cervical squamous cell carcinoma, albeit at a low frequency. In conclusion, the present study identified three potentially pathogenic ESR1 mutations in Chinese cervical squamous cell carcinoma samples, but not in other subtypes.