Introduction
The normal development and proliferation of breast epithelium are regulated by estrogen, which acts as the primary driver of pubertal growth, and by progesterone, which directs the expansion of the adult mammary epithelium.1 The historical link between these hormones and malignancy has been recognized for over a century.2 Jensen’s 1967 characterization of the estrogen receptor (ER) protein fundamentally changed the understanding of hormone function and established the ER as a molecular target for endocrine therapy (ET).3 Approximately 75% of breast cancers are hormone receptor-positive, and those patients benefit from ET, such as the traditional selective estrogen receptor modulator (SERM) tamoxifen and aromatase inhibitors, which effectively block these signaling pathways.4,5
With the advent of new-generation SERMs, selective estrogen receptor degraders (SERDs), and cyclin-dependent kinase 4/6 (CDK4/6) inhibitors, recent clinical trials such as SERENA-6, VERITAC-2, and CAPItello-291, as well as the ongoing SERENA-4 trial, represent a major shift in the treatment approach for hormone receptor-positive/HER2-negative advanced and metastatic breast cancer. Collectively, these trials reflect a shift toward more personalized, biomarker-driven therapeutic strategies that improve clinical outcomes in defined patient populations.6-8ESR1 mutations are associated with metastatic disease progression and worse clinical outcomes. Real-time testing for ESR1 mutations using circulating tumor DNA (ctDNA)-based liquid biopsy at disease progression is increasingly used to guide treatment decisions.
As breast pathology advances and new diagnostic modalities emerge, pathologists are taking on expanded roles. By helping clinicians interpret molecular testing results and identify molecular biomarkers, pathologists are becoming increasingly involved in the era of precision medicine. This mini review highlights recent developments in ESR1 testing and their clinical implications, with a focus on the practical challenges of implementing and interpreting ESR1 testing as targeted therapies become more widely integrated into breast cancer care.
ESR1 and the ER signaling pathway
Hormone receptor-positive/HER2-negative breast cancer is the most prevalent molecular subtype of breast cancer. Estrogen receptor alpha (ERα), encoded by the ESR1 gene, acts as a major oncogenic driver and therapeutic target in approximately 75% of breast cancer cases.9 Estrogen binding to ER induces receptor dimerization and nuclear translocation, followed by binding to estrogen response elements and transcription of target genes responsible for cell proliferation, survival, and differentiation. ER signaling is not limited to direct DNA binding; it also interacts bidirectionally with kinase pathways, including the PI3K-AKT-mTOR and RAS-RAF-MEK pathways: ER can activate kinase signaling, and kinases can phosphorylate ER to promote ligand-independent transcription (Fig. 1).10 This cross-talk is a major mechanism of drug resistance in hormone receptor-positive breast cancer.
Next-generation endocrine therapies
Endocrine therapy (ET) has long been used to treat breast cancer. The traditional SERM tamoxifen, approved by the U.S. Food and Drug Administration (FDA) in 1977, competitively binds to estrogen receptors, blocking ER signaling.11 The first-generation SERD fulvestrant, approved by the FDA in 2002, requires intramuscular injections. Fulvestrant can bind to the ER, induce structural changes that destabilize the protein complex, and trigger ER degradation.12
Patients with advanced or metastatic hormone receptor-positive breast cancer can develop endocrine resistance because of acquired somatic ESR1 mutations; next-generation therapies have been developed to overcome this resistance. Agents in three main therapeutic classes have received FDA approval or fast-track designation in recent years: oral SERDs, SERMs, and proteolysis-targeting chimeras (PROTACs).13,14 Additional novel drug classes include complete estrogen receptor antagonists. Selective estrogen receptor covalent antagonists are also under active investigation.13
SERMs work by competing with endogenous estrogen for binding to the ER, while leaving the receptor intact. SERDs are considered pure antagonists. When a SERD binds to the ER, it induces a receptor conformational change, impairing dimerization and nuclear translocation and promoting degradation through the ubiquitin-proteasome system. PROTACs represent a newer targeted protein-degradation strategy. A PROTAC molecule, functioning as a heterobifunctional protein degrader, is designed to bind to the ER at one end and an E3 ubiquitin ligase at the other. By bringing either wild-type or mutant ER into proximity with the E3 ligase, a PROTAC promotes ER ubiquitination and proteasomal degradation.15
The FDA approved the SERD elacestrant (Orserdu, Stemline Therapeutics, Inc.) in 2023 for postmenopausal women or adult men, the SERD imlunestrant (Inluriyo, Eli Lilly and Company) in 2025, and the PROTAC vepdegestrant (Veppanu, Arvinas Operations, Inc.) in 2026 for adults with ER-positive/HER2-negative, ESR1-mutated advanced or metastatic breast cancer with disease progression following at least one line of ET. These approvals were based on the EMERALD (NCT03778931), EMBER-3 (NCT04975308), and VERITAC-2 (NCT05654623), respectively.6,16-20 The next-generation SERDs, SERMs, and PROTACs that are FDA-approved or under active investigation in late-phase trials are summarized in Table 1. In addition, the SERM lasofoxifene has been granted fast-track designation by the FDA for patients with hormone receptor-positive/HER2-negative metastatic breast cancer with ESR1 mutations.21,22 Camizestrant (Etcamah, AstraZeneca) received FDA accelerated approval in September 2026 in combination with a CDK4/6 inhibitor for adults with hormone receptor-positive/HER2-negative locally advanced or metastatic breast cancer upon detection of an ESR1 mutation during aromatase inhibitor and CDK4/6 inhibitor therapy, based on an FDA-authorized test.23 Giredestrant remains under phase 3 evaluation.
| Drug name | Drug class | Study | FDA status |
|---|
| Elacestrant | SERD | EMERALD (NCT03778931) | Approved 2023 |
| Imlunestrant | SERD | EMBER-3 (NCT04975308) | Approved 2025 |
| Camizestrant | SERD | SERENA-6 (NCT04964934) | Accelerated approval 2026 |
| Giredestrant | SERD | persevERA (NCT04546009) | Not yet approved |
| Lasofoxifene | SERM | ELAINE 1 (NCT03781063); ELAINE 2 (NCT04432454) | Fast-track designation |
| Vepdegestrant | PROTAC | VERITAC-2 (NCT05654623) | Approved 2026 |
For elacestrant, imlunestrant, vepdegestrant, and camizestrant, the FDA has approved the Guardant360 CDx liquid biopsy assay as a companion diagnostic to identify patients with ESR1 mutations for the corresponding treatment indications.16,17,20,23 This highlights the growing importance of liquid biopsy testing in contemporary breast cancer management.
ctDNA testing for ESR1 mutation detection
The recent approvals of next-generation endocrine therapies together with the approval of the Guardant360 CDx assay as a companion diagnostic reflect an increasing emphasis on precision medicine in endocrine-resistant breast cancer. Because these therapies are indicated for patients whose tumors harbor ESR1 mutations, their appropriate use depends on the timely and accurate detection of these actionable ESR1 mutations.
ESR1 alterations, specifically missense mutations in the ligand-binding domain (LBD) of the ER protein, are recognized as a major acquired resistance mechanism in patients with hormone receptor-positive/HER2-negative metastatic breast cancer receiving ET.24,25 Residues E380, S463, V534, Y537, and D538 are recurrently altered, with Y537S, Y537N, Y537C, and D538G among the hotspot variants. These ESR1 LBD mutations stabilize the active conformation of helix 12, allowing transcriptional activity even when estrogen levels are suppressed by aromatase inhibition, and result in constitutive activation of the ER pathway independent of estrogen.26
ESR1 mutations are uncommon in untreated primary breast cancer but become substantially enriched in metastatic hormone receptor-positive/HER2-negative disease after ET. ESR1 mutations are more frequently observed in treated metastatic disease (36%) than in the adjuvant setting (6%) or ET-naïve metastatic breast cancer (< 1%).27 Testing primary tumor tissue may yield more negative results and may not reflect tumor evolution during treatment. Therefore, testing for ESR1 mutations in blood samples collected during treatment may provide contemporaneous tumor information to guide treatment decisions.28 ctDNA-based liquid biopsy testing can help identify patients who are not responding to treatment or who are at higher risk of relapse. Studies indicate that ctDNA may track treatment response and help predict recurrence in early-stage disease.29,30 Plasma ctDNA has shown higher detection rates for ESR1 mutations than tissue testing in some studies.31
Liquid biopsy, a minimally invasive method for monitoring tumor recurrence, is increasingly used in clinical oncology. Cell-free DNA extracted from blood and containing tumor-derived ctDNA offers a less invasive option than tissue biopsy. Two major molecular assay designs are used in clinical practice. Targeted next-generation sequencing (NGS) panels can detect ESR1 mutations along with alterations in other important genes, such as PIK3CA, AKT1, PTEN, and ERBB2, thereby supporting broader therapeutic planning and identifying additional actionable biomarkers. Highly sensitive polymerase chain reaction-based approaches, including droplet digital polymerase chain reaction, can efficiently interrogate known ESR1 hotspot variants but provide less comprehensive genomic profiling. The clinical utility of liquid biopsy was demonstrated in the PADA-1 trial, where multiplex droplet digital polymerase chain reaction was employed for serial ESR1 mutation monitoring and a preemptive treatment switch upon detection of an ESR1 mutation significantly improved progression-free survival.32 Similarly, in the SERENA-6 trial, switching to camizestrant plus a CDK4/6 inhibitor when an ESR1 mutation was detected by NGS prolonged progression-free survival.33 On the basis of findings across multiple studies, guidelines now recommend ctDNA testing for ESR1 mutations in patients with hormone receptor-positive/HER2-negative metastatic breast cancer.34,35 As the cost of NGS has decreased in recent years, comprehensive NGS profiling has become a more widely used strategy and can assess an increasing number of biomarkers (Fig. 2).
Nevertheless, although liquid biopsy can support disease monitoring and recurrence detection, it has important technical limitations. It may be less sensitive than tissue-based NGS when a blood sample contains a low tumor fraction, whereas a tissue sample may be more enriched in tumor material. Fluctuations in ctDNA levels may correlate with the course of the disease; a higher tumor fraction can reflect greater tumor DNA shedding and has been associated with higher tumor burden, metastatic disease, and poorer prognosis.36 Consequently, liquid biopsy specimens collected at different time points can have variable ctDNA levels and assay results. A low tumor fraction increases the risk of a false-negative result and of missing an acquired ESR1 mutation because of limited tumor material in the specimen. ctDNA test results from specimens with low tumor fractions should be interpreted with caution. In addition, NGS-based ctDNA testing varies across clinical laboratories in sequencing depth, gene panel size, and alteration-specific detection thresholds.
In summary, while highly sensitive digital polymerase chain reaction and comprehensive NGS panels have made ctDNA testing a useful tool for longitudinal disease monitoring and early resistance detection, the approach retains important limitations. A key limitation is the variability of tumor fractions in the blood, where low ctDNA shedding can cause false-negative results that obscure ESR1 mutations. Guidelines from the National Comprehensive Cancer Network recommend careful interpretation of low-fraction samples and repeat liquid biopsy or tissue biopsy to ensure accurate therapeutic planning.37
ESR1 genomic alterations beyond hotspot missense mutations
Although hotspot LBD point mutations are the best-established clinically actionable ESR1 alterations, the clinical significance of non-hotspot ESR1 alterations remains uncertain, and comparative data on their effects on treatment resistance are limited. In addition, other types of alterations, such as ESR1 copy-number loss, amplification, and fusion, have also been detected in endocrine-resistant metastatic breast cancer but are less well studied.38
ESR1 amplification, reported in approximately 5–20% of hormone receptor-positive breast cancers by different studies, results in increased ER expression and enhanced estrogen signaling.25 While ESR1 amplification may contribute to endocrine sensitivity in some tumors, it has also been associated with adaptive resistance through persistent ER pathway activation despite estrogen deprivation. The clinical significance of ESR1 amplifications in early-stage and recurrent disease is still under investigation.39
ESR1 fusions have been estimated to occur in 1–10% of hormone receptor-positive breast cancers by different studies.40 Recurrent fusions often contain the first 1–6 exons of the ESR1 gene, which encode the activation-function and DNA-binding domains but lack the ligand-binding domain and are fused in frame to various 3′ partner genes. These fusion proteins can be constitutively active and ligand-independent, and the loss of the ligand-binding domain may make tumors less sensitive to therapies that require binding to the ER LBD.41
Although these alterations are relatively rare compared with ESR1 missense mutations and may be less readily detected by early-generation liquid biopsy assays, their reported detection is increasing as comprehensive NGS assays incorporating copy-number analysis and RNA sequencing for fusions are used more frequently in metastatic settings. Unlike ESR1 hotspot mutations, which currently guide the use of next-generation SERDs, SERMs, and PROTACs, the clinical significance of ESR1 amplifications and fusions is not yet fully understood and is under active investigation.
The evolving role of pathologists in ESR1 testing
Recent advances and emerging therapies, together with guidelines recommending ESR1 mutation testing at recurrence or progression, emphasize the evolving role of pathologists within the multidisciplinary breast cancer team. In the era of precision medicine, pathologists contribute to personalized patient care through diagnosis and by guiding molecular testing strategies and interpreting complex biomolecular data. The role of pathologists has expanded beyond diagnosis to broader participation throughout the clinical care pathway. By understanding ER signaling, ESR1 biology, and the application of ESR1 mutation testing, including liquid biopsy, pathologists’ responsibilities now include supporting decisions about the optimal timing and modality for ESR1 mutation testing, advising on plasma versus tissue sample selection, and facilitating communication of molecular test results within the care team.
Limitations
As a mini review, the major limitations include its narrative approach and the potential for literature-selection bias. The rapid evolution of evidence and regulatory guidance in this area may mean that some recent advances are not fully captured. Additionally, heterogeneity among ctDNA testing platforms and the relatively limited data on non-hotspot ESR1 alterations may restrict the generalizability and comprehensiveness of the conclusions drawn. Future systematic reviews and meta-analyses could strengthen the evidence base and further clarify the clinical utility of ESR1 testing.
Conclusions
ERα signaling is a key driver of the development and progression of hormone receptor-positive/HER2-negative breast cancers, while acquired ESR1 alterations are a major mechanism of endocrine resistance. Liquid biopsy is now an established clinical approach to ESR1 testing in advanced or metastatic hormone receptor-positive/HER2-negative breast cancer, including at recurrence or progression during endocrine therapy and, in selected patients, during ongoing aromatase inhibitor plus CDK4/6 inhibitor therapy to detect emerging ESR1 mutations. It is also being investigated for recurrence prediction and treatment monitoring. However, the regulatory landscape for liquid biopsies and targeted therapies is evolving rapidly. As new targeted therapies and companion diagnostics are developed, the role of liquid biopsy may continue to expand. Pathologists should remain current with advances relevant to their supporting role in multidisciplinary breast cancer care.
Declarations
Funding
No funding was received for this review.
Conflict of interest
YW is an Editorial Board Member of the Journal of Clinical and Translational Pathology. LL has no conflicts of interest to disclose.
Author contributions
Drafting the manuscript (LL, YW); critical revision of the manuscript (LL, YW). Both authors made significant contributions to this review and approved the final manuscript.