Abstract: Precision medicine has reshaped the management of advanced hormone receptor–positive, human epidermal growth factor receptor 2–negative breast cancer by coupling treatment with tumor genomics rather than clinicopathologic features alone. In this review, we highlight the role of next-generation sequencing (NGS) in our clinical practice. We summarize the performance characteristics of various NGS platforms and outline the available techniques of NGS. We also describe clinically relevant genomic alterations and targeted treatments.
Introduction
Breast cancer is the most common cancer diagnosed among women in the United States, with an estimated 310,720 new cases in 2024.1 Despite this increase in the incidence of breast cancer, the mortality rate has decreased by 44% from 1989 to 2022.2 Hormone receptor–positive (HR+), human epidermal growth factor receptor 2–negative (HER2–) breast cancer is the most common subtype of breast cancer.2 The development of a genomic test called the Prediction Analysis of Microarray of 50 genes (PAM 50) assay by Perou and colleagues led to the first molecular classification of breast cancer, which started the era of molecular genomics in breast cancer. It characterizes breast cancer as luminal A (HR+, HER2–, and low proliferation rates), luminal B (HR+, HER2–, higher proliferation rates than luminal A), HER2-enriched (HR+, HER2+, and high proliferation rates), basal-like (HR–, HER2–, and high proliferation rates), and normal-like (similar to normal breast tissue, with low proliferation rates). This stratification so far is used in the prognostic evaluation of breast cancer, with limited use in therapy planning.3,4
Since the development of PAM 50, genomic profiling has become an integral part of the management of advanced breast cancer, and here we aim to review the integration of next-generation sequencing (NGS) in the management of HR+ advanced breast cancer.
Next-Generation Sequencing
It took nearly a quarter of a century to develop DNA sequencing technology after its discovery in 1953.5,6 Sanger sequencing, also known as first-generation sequencing, used a “sequencing by synthesis” approach to determine nucleic acid sequences one nucleotide at a time. After automation by Applied Biosystems, this approach was used in the Human Genome Project.7 However, the limited throughput of first-generation sequencing led to the development of NGS, which enables massive parallel sequencing and greatly increased data output.7 NGS has since driven major advances in cancer research through the precise characterization of cancer genomes and epigenomes.8
Various technologies under the umbrella of NGS have been adopted for nucleic acid sequencing in cancer genomics. Whole-genome sequencing (WGS) provides a comprehensive analysis of the entire genome, including coding and noncoding regions. In contrast, whole-exome sequencing (WES) focuses on the protein-coding regions only, which constitute approximately 1% to 2% of the genome. This approach captures approximately 85% of known disease-causing mutations, including most oncogenic driver alterations.9,10 These techniques have been applied in early diagnosis, analysis of recurrence, treatment, and research.11 WES may miss important biomarkers like the complex mutational signatures of homologous recombination deficiency, and it provides less accurate estimates of tumor mutational burden (TMB) than WGS, particularly in low-TMB tumors.12,13
Because WGS and WES remain largely cost-prohibitive, focus has shifted to targeted gene panels (TGPs), which are focused and affordable.14-16 In a study by Frampton and colleagues, TGPs identified clinically actionable mutations in 76% of 2221 tumors, a 3-fold higher rate of detection in comparison with other techniques.17
Liquid biopsy, which is a noninvasive tool for examining body fluids, is increasingly being used to detect biomarkers for disease monitoring and for improving diagnosis and treatment.18 Various biomarkers in the physiologic fluids can be examined, including circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), cell-free DNA (cfDNA), and circulating exomes.19 TGPs employing liquid biopsy are increasingly utilized in oncology for therapy selection, treatment monitoring, and measurable residual disease detection in patients with advanced solid malignancies, particularly when tissue samples are unavailable or insufficient.20-22
Tissue-Based NGS
In 2017, the US Food and Drug Administration (FDA) approved multiple NGS-based assays for use in oncology.23,24 These assays enable comprehensive genomic profiling by analyzing hundreds of cancer-related genes in solid tumors with the use of hybrid capture–based enrichment methods.25 Some of the platforms have also been approved as companion diagnostics for targeted therapies and immunotherapies, supporting their role in guiding treatment selection for patients with advanced and recurrent cancers. Since then, additional NGS assays with varying gene coverage, analytical capabilities, and clinical applications have been developed and approved, reflecting the rapid evolution of genomic testing in oncology.
Real-world data and clinical trials have demonstrated that NGS-guided therapy changes management in a clinically meaningful subset of patients. In the SAFIR02-BREAST trial, patients who received targeted treatment had better progression-free survival (PFS; adjusted hazard ratio [HR], 0.41; 90% CI, 0.27-0.61; P<.001) if they had level 1 or 2 mutations on the European Society for Medical Oncology (ESMO) Scale for Clinical Actionability of molecular Targets (ESCAT). The improvement with targeted therapy was especially pronounced among patients who had germline BRCA1/2 mutations.26 Various studies with real-world data have shown that NGS is helpful in recognizing both predictive biomarkers and drug-targetable markers, the most common being PIK3CA.27-29 NGS has also been instrumental in identifying resistance mutations, such as ESR1 mutations, that arise after prolonged antiestrogen therapy. These mutations typically occur in the ligand-binding domain and lead to the constitutive activation of estrogen receptor (ER) signaling.30 NGS testing of patients who have been exposed to cyclin-dependent kinase 4/6 (CDK4/6) inhibitors revealed resistance mutations such as RB1 loss, activating alterations of AKT1, RAS, AURKA, CCNE2, ERBB2, and FGFR2, and loss of ER expression that can affect cell cycle regulatory proteins as well as signaling pathways.31 The presence of these mutations provides an opportunity for research and the development of new therapeutics. Various novel therapies are in development that aim to overcome resistance mechanisms, such as by inhibiting CHEK1 and AURKA or by adding mitogen-activated protein kinase (MAPK) pathway inhibitors to CDK4/6 inhibitors.31-33 Preclinical studies have shown that CDK2 inhibition can lead to the resensitization of breast cancer cells to CDK4/6 inhibition, as upregulation of CDK2 is one of the mechanisms of resistance to CDK4/6 inhibition.34 Various ongoing trials of CDK2-inhibiting drugs, both as monotherapy and as part of combination treatment, especially after the progression of cancer in patients on CDK4/6 inhibition, are in the recruitment phase.34
Unlike DNA sequencing, which provides information on genomic alterations, RNA sequencing provides dynamic information about which genes are actively transcribed, offering insights into cellular function and disease mechanisms.35 It is used to characterize the tumor microenvironment, infer immune cell populations, and analyze gene expression signatures relevant to diagnosis, prognosis, and therapeutic response.36,37 In addition, RNA sequencing detects more actionable gene fusions than DNA-based methods do, particularly in non–small cell lung cancer, sarcomas, and low-grade gliomas.38-40 In breast cancer, RNA sequencing aids in the quantification of ESR1 messenger RNA.41,42
Liquid NGS
Liquid NGS provides a noninvasive tool for genomic profiling. Various types of liquid NGS biomarkers can be tested. CTCs are used primarily in HR+ breast cancer as a predictive and prognostic marker. The number of CTCs is associated with prognosis, with higher CTC counts being linked to poorer prognosis. In addition, a single positive CTC assay result after the completion of treatment is predictive of late clinical recurrence.43-45
The monitoring of ctDNA has been utilized to evaluate therapy response, with rising ctDNA levels being associated with progression.46 Research also suggests that personalized ctDNA monitoring can make it possible to decrease imaging frequency.46 In the SOLAR-1 trial, ctDNA was used to determine PFS.47 Studies have shown a concordance rate of up to 90% between blood-based and tissue-based NGS.48-50 Quantitative ctDNA testing helps to monitor disease, and a rise in ctDNA points toward progression. In addition, ctDNA is helpful in identifying early genomic alterations. For example, ESR1 and PIK3CA mutations can be detected early with ctDNA.51,52 The SERENA-6 trial demonstrated the usefulness of ctDNA in the early detection of ESR1 mutations, which led to a switch in therapy to camizestrant and subsequently improved PFS.53 In the NCT05826964 trial, ctDNA will be tested frequently in patients with HR+/HER2– metastatic breast cancer (MBC) who are on an aromatase inhibitor (AI) and a CDK4/6 inhibitor, with the goal of switching therapy in cases of early molecular progression. The MONDRIAN study (NCT04720729) is evaluating the use of ctDNA measurement to determine if a treatment regimen should be changed in patients with HER2+ MBC who are receiving chemotherapy.
Although liquid NGS also has the benefits of being a minimally invasive test, able to capture tumor heterogeneity and aid in real-time disease monitoring with a quick turnaround time, it has limitations as well.54-58 Its sensitivity is lower than that of tissue-based NGS, and the false-negative rate is higher owing to low ctDNA levels. Liquid NGS may also produce results that do not agree with those of tissue-based NGS, as liquid NGS may detect variants not found in a tissue sample.59-62 In addition, tissue-based NGS remains the standard for TMB and microsatellite instability testing. Furthermore, liquid NGS cannot be used for ancillary studies (eg, in situ hybridization, RNA-based fusion detection, molecular subtyping) that require an intact cellular structure (Table 1).59-61,63
Clinical Implications of Specific Genomic Alterations
Endocrine-based therapy is the guideline-preferred first-line treatment for HR+ MBC in the absence of visceral crisis, with endocrine therapy plus a CDK4/6 inhibitor the standard in most patients.85-88 Ribociclib (Kisqali, Novartis), abemaciclib (Verzenio, Lilly), and palbociclib (Ibrance, Pfizer) have all shown substantial PFS and overall survival (OS) benefit when combined with an AI or fulvestrant and are now widely used in the first-line setting.89-94
Despite these advances, the optimal sequencing after progression in patients on CDK4/6 inhibition remains unsettled, and overcoming endocrine resistance is a major challenge. Primary endocrine resistance is relapse within the initial 2 years after adjuvant endocrine therapy or progression within the first 6 months after endocrine therapy for MBC; secondary resistance is relapse after at least 2 years of adjuvant endocrine therapy, or relapse during or within 1 year after the completion of adjuvant treatment for early breast cancer, or progression after at least 6 months following the initiation of endocrine therapy for advanced/metastatic breast cancer.95 Multiple mechanisms drive resistance, including alterations in ER signaling and cross talk with growth factor pathways such as phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR).96
PI3K/AKT/mTOR Signaling Pathway Activation
The PIK3CA mutation either can occur in early tumor development or can be an acquired mutation at the time of disease progression.97 PIK3CA mutations occur in roughly 40% of ER+/HER2– breast cancers and lead to activation of the PI3K/AKT/mTOR signaling pathway; phosphatase and tensin homologue (PTEN) loss of AKT mutations can similarly promote pathway overactivity.98,99 PI3K activation generates phosphatidylinositol 3,4,5-triphosphate with downstream activation of AKT and MTOR, whereas PTEN counteracts this by dephosphorylating pathway intermediates.99,100
Alpelisib (Piqray, Novartis), an orally bioavailable selective inhibitor of PIK3CA, improved PFS (11 vs 5.7 months; HR, 0.65; 95% CI, 0.50-0.85; P<.001) and response rates when combined with fulvestrant in PIK3CA-mutant HR+/HER2– MBC in the SOLAR-1 trial, although grade 3 or higher hyperglycemia (36.6%), rash, and diarrhea were common and led to a 25% discontinuation rate.47 OS was numerically but not statistically significantly higher with alpelisib plus fulvestrant than with fulvestrant alone, at 39.3 vs 31.4 months, respectively (HR, 0.86; 95% CI, 0.64-1.15; P=.15).47 A key limitation was minimal prior CDK4/6 inhibitor exposure. In the phase 2 BYLieve study, 50.4% of patients remained progression-free at 6 months on alpelisib/fulvestrant after progression on a CDK4/6 inhibitor, supporting its activity in this setting.101
The oral pan-AKT inhibitor capivasertib (Truqap, AstraZeneca) doubled median PFS from 3.6 to 7.2 months overall when added to fulvestrant in the CAPItello-291 trial (HR, 0.60; 95% CI, 0.51-0.71) in patients with MBC whose disease progressed on an AI with or without a CDK4/6 inhibitor.99 OS trends favored capivasertib without reaching significance at interim analysis, and benefit in AKT pathway wild-type disease was modest (HR, 0.79; 95% CI, 0.61-1.02), consistent with prior FAKTION data suggesting maximal benefit in AKT1/PTEN/PIK3CA-altered tumors.99,102 Regulatory approval is therefore restricted to this biomarker-defined population, and the safety profile, with rates of high-grade hyperglycemia much lower than with alpelisib, is comparatively more manageable.47,99
Inavolisib (Itovebi, Genentech), a potent selective inhibitor of the alpha isoform of the p110 catalytic subunit of the PI3K complex, improved median PFS from 7.3 to 15 months (HR, 0.43; 95% CI, 0.32-0.59) and the overall response rate (ORR; 58.4% vs 25%) when added to palbociclib/fulvestrant in PIK3CA-mutated HR+/HER2– MBC in INAVO120, with better interim OS (34 vs 27 months; HR, 0.67; 95% CI, 0.48-0.94) but increased rates of hyperglycemia, stomatitis, and diarrhea.103,104 Data with other CDK4/6 inhibitors and after adjuvant CDK4/6 exposure remain limited.
Gedatolisib (Gedatolisib, Celcuity), a class I pan-PI3K and dual mTORC1/2 inhibitor, improved PFS in the phase 3 VIKTORIA-1 trial in PIK3CA wild-type disease. VIKTORIA-1 was a global, open-label, 2-part clinical trial that included study 1 (PIK3CA wild-type patients) and study 2 (PIK3CA-mutated patients), with results available only for study 1. The researchers found that in the PIK3CA wild-type cohort, median PFS was 9.3 months with gedatolisib/palbociclib/fulvestrant and 7.4 months with gedatolisib/fulvestrant vs 2 months with fulvestrant alone (HRs, 0.24 and 0.33, respectively), supporting multitarget PI3K/mTOR strategies beyond PIK3CA-mutant tumors.105
For patients without targetable PIK3CA/AKT/PTEN or ESR1 alterations after an AI plus a CDK4/6 inhibitor, mTOR inhibition remains an option. Everolimus combined with endocrine therapy increased time to progression and PFS in GINECO (tamoxifen + everolimus), BOLERO-2 (everolimus + exemestane), and PrE0102 (everolimus + fulvestrant), although all preceded widespread CDK4/6 inhibitor use.106-108 A prospective study by Vasseur and colleagues supports everolimus-plus-fulvestrant activity after a CDK4/6 inhibitor.109 The dual mTORC1/2 inhibitor vistusertib did not outperform everolimus in the MANTA study, likely reflecting incomplete mTORC1 inhibition or the limited clinical impact of mTORC2.110
ESR1 Mutations
ESR1 mutations confer ligand-independent ER activation and are a key mechanism of resistance to AIs, although selective ER degraders (SERDs) and some selective ER modulators (SERMs) can partially overcome this problem.111 Mutation rates range from approximately 1% in patients with treatment-naive disease to nearly 40% in heavily pretreated patients, and mutations frequently emerge in patients on endocrine therapy, prompting recommendations for testing at progression with tissue or ctDNA, with ctDNA preferred for sensitivity and dynamic monitoring.112-116
Elacestrant (Orserdu, Stemline Therapeutics), an oral SERD, significantly improved PFS vs standard endocrine monotherapy in the phase 3 EMERALD trial, both overall (HR, 0.70; 95% CI, 0.55-0.88) and in patients with ESR1-mutant tumors (HR, 0.55; 95% CI, 0.39-0.77), with a 12-month PFS rate of 26.8% vs 8.2% in the ESR1-mutant subgroup and more pronounced benefit in patients with at least 12 months of prior CDK4/6-endocrine therapy (median PFS, 8.6 vs 1.9 months; HR, 0.41; 95% CI, 0.26-0.63).117,118 An ongoing umbrella trial called ELEVATE is exploring elacestrant in combination with agents such as everolimus, abemaciclib, and alpelisib; early data on everolimus and abemaciclib show consistent PFS benefit regardless of ESR1 mutation status after progression on endocrine therapy with or without a CDK4/6 inhibitor.119
In the PADA-1 trial, among patients on an AI plus palbociclib in whom an ESR1 mutation developed on ctDNA without radiologic progression, switching from an AI to fulvestrant while continuing palbociclib significantly prolonged PFS. This finding supports a ctDNA-guided, preprogression switch strategy, although further prospective validation is required before routine adoption.120 SERENA-6 extended this concept by randomizing patients with an emergent ESR1 mutation on first-line treatment with an AI plus a CDK4/6 inhibitor (without clinical progression) either to continue current therapy or to switch to camizestrant, a next-generation SERD, with the same CDK4/6 inhibitor. Camizestrant nearly doubled PFS (16 vs 9.2 months; HR, 0.44; 95% CI, 0.31-0.60), with OS data pending.53
Imlunestrant (Inluriyo, Lilly), another next-generation oral SERD, demonstrated PFS improvement vs standard endocrine therapy in ESR1-mutant disease (5.5 vs 3.8 months; P<.001) in the EMBER-3 trial but not in the overall population. The combination of imlunestrant plus abemaciclib improved PFS to 9.4 months vs 5.5 months with imlunestrant alone.121 Rates of central nervous system progression were lower with imlunestrant than with standard of care in patients with ESR1-mutant disease (1.5% vs 6.7%; HR, 0.18; 95% CI, 0.04-0.90) and in the overall population (1.6% vs 3.0%; HR, 0.47; 95% CI, 0.16-1.38).121 Updated results showed an OS benefit for imlunestrant vs standard endocrine therapy in ESR1-mutant cases (34.5 vs 23.1 months; HR, 0.60; P=.0043), although OS data in the overall population remain immature, particularly for the imlunestrant/abemaciclib combination.122
Given increasing early-line CDK4/6 inhibitor use, post-CDK4/6 strategies are essential. The evERA trial evaluated giredestrant plus everolimus vs physician’s choice of endocrine therapy plus everolimus in ER+/HER2– MBC after CDK4/6 inhibitor and showed improved PFS in ESR1-mutant (9.99 vs 5.45 months; HR, 0.38; 95% CI, 0.27-0.54) and overall populations (8.77 vs 5.49 months; HR, 0.56; 95% CI, 0.44-0.71), with benefit irrespective of PIK3CA/AKT1/PTEN status and prior CDK4/6 inhibitor duration.123,124
BRCA1/2 and PALB2 Mutations
Approximately 5% of patients with breast cancer harbor germline BRCA mutations, with a higher prevalence in HER2– subtypes. Poly(ADP-ribose) polymerase inhibitors are now standard options for germline BRCA-associated advanced disease.125 In OlympiAD, olaparib (Lynparza, AstraZeneca) improved PFS vs nonplatinum chemotherapy (7 vs 4.2 months; HR, 0.58; 95% CI, 0.43-0.80), with a trend toward greater benefit in triple-negative disease and a more modest effect in HR+ tumors (HR, 0.82; 95% CI, 0.55-1.26). OS was not significantly different overall, but a prespecified subgroup without prior chemotherapy for metastatic disease derived an OS benefit (HR, 0.51; 95% CI, 0.29-0.90).126,127 Talazoparib (Talzenna, Pfizer) in EMBRACA similarly improved PFS (8.6 vs 5.6 months; HR, 0.54; 95% CI, 0.41-0.71) in germline BRCA-mutated HER2– disease, with benefit maintained in patients with HR+ disease and no significant OS difference.128,129 Because PARP inhibitors are generally better tolerated than many chemotherapies and lack cumulative toxicity, guidelines recommend their use after progression on endocrine therapy in germline BRCA-mutated, HR+ MBC, although the best sequencing relative to CDK4/6 inhibition is unclear. In addition, retrospective data suggest shorter PFS and OS with CDK4/6 inhibition in germline BRCA-mutated HR+ disease.85,130,131
Beyond BRCA, phase 2 data from the TBCRC 048 study support olaparib activity in cases with other homologous recombination repair defects; high response rates and prolonged PFS were observed in germline PALB2-mutated MBC (ORR, ~75%-82%; PFS, ~9.6-13.3 months), with meaningful activity in somatic BRCA1/2 mutations and minimal benefit in ATM- or CHEK2-mutated tumors.132,133
HER2-Low and HER2 Mutations
HER+ disease is defined by an immunochemistry (IHC) score of 3+ or of 2+ with in situ hybridization amplification. HER2– tumors may express low levels of HER2 (HER2-low), which is now an actionable category.134,135 DESTINY-Breast04 established trastuzumab deruxtecan (Enhertu, Daiichi-Sankyo/AstraZeneca) as superior to chemotherapy in HER2-low MBC previously treated with endocrine therapy and 1 to 2 lines of chemotherapy, improving PFS (10.1 vs 5.4 months; HR, 0.51; 95% CI, 0.40-0.64) and OS (23.9 vs 17.5 months; HR, 0.64; 95% CI, 0.49-0.84) in the HR+ cohort.136 DESTINY-Breast06 extended these findings to earlier-line treatment and to HER2-ultralow (IHC of 0 with faint membrane staining [0<IHC<1]) tumors. The trial showed PFS benefit in HER2-low (13.2 vs 8.1 months; HR, 0.64) and similar gains in the intention-to-treat population, suggesting extension of benefit to HER2-ultralow disease, leading to earlier-line approval in HR+ HER2-low/ultralow disease, and supporting more refined scoring of IHC 0 cases by membrane staining.137,138
HER2-activating mutations, distinct from amplification, occur in approximately 3% to 5% of HER2-nonamplified breast cancers and up to 10% of lobular cancers, and they represent another targetable subset.139,140 In the phase 2 MutHER trial, neratinib (Nerlynx, Puma Biotechnology) plus fulvestrant achieved clinical benefit rates of 30% to 38% in HR+/HER2-mutant, HER2-nonamplified MBC, regardless of prior CDK4/6 inhibition.139 SUMMIT showed that neratinib, trastuzumab, and fulvestrant produced a 39% ORR and median PFS of 8.3 months in HR+/HER2-mutant disease after CDK4/6 inhibition.141 A basket study of tucatinib (Tukysa, Seagen) plus trastuzumab (±fulvestrant) reported an ORR of 41.9%, median duration of response 12.6 months, and PFS of 9.5 months, collectively supporting HER2-targeted combinations in this genomically defined group (Table 2).142
Challenges and Future Implications
Despite its transformative role, NGS faces barriers that include high cost, limited access, and low patient awareness. Socioeconomic disparities, geographic constraints, and variability in the healthcare infrastructure further restrict equitable utilization.143,144 Tumor heterogeneity in breast cancer raises concerns about the representativeness of small biopsy samples, and the lack of standardization of the NGS technique and the variability in testing technique make profiling challenging.
Tissue sampling following progression is often difficult. The emergence of ctDNA has offered minimally invasive alternatives for detecting targetable mutations, monitoring resistance, and assessing prognosis. The integration of artificial intelligence remains limited, but alternative approaches to biomarker evaluation are under research, such as mass spectrometry–based proteomics, that enable the detection of proteins like CDK1, the status of ERBB2 and the ER, and tumor grading.145,146
Nanoparticles have been studied and utilized for the precise delivery of drug or contrast media to tumor cells, limiting the exposure of noncancer cells and decreasing toxicity.147 A study by Pourtau and colleagues showed that trastuzumab conjugated with a nanoparticle delivery system can specifically target bone metastasis.148 More recently, the use of an antibody-conjugated, tumor-targeted nanolipogel to deliver CRISPR/Cas9 plasmids into triple-negative breast cancer for genomic editing led to the knockout of lipocalin 2 and resulted in the significant suppression of tumor growth.149 In ER+ breast cells, estradiol-conjugated nanogels and polymerases have demonstrated enhanced uptake with cytotoxic effects.150 The concomitant delivery of fulvestrant and abemaciclib has been shown to improve drug half-life and bioavailability.151
Proteolysis targeting chimera (PROTAC) technology enables targeted protein degradation via the ubiquitin-proteasome system. In the VERITAC trial, vepdegestrant (Veppanu, Arvinas) improved PFS more effectively than fulvestrant in patients with ESR1-mutant HR+/HER2– MBC whose disease progressed on prior endocrine therapy including a CDK4/6 inhibitor (5.0 vs 3.7 months; HR, 0.58), with modest benefit in the overall population.152,153
CDK2 has emerged as a key driver of resistance to CDK4/6 inhibition, primarily through CCNE1 amplification and cyclin E1 overexpression, which maintain G1/S cell cycle progression despite CDK4/6 blockade.154 Several selective CDK2 inhibitors are in early clinical development. For example, tegtociclib (PF-07104091) is being evaluated as monotherapy and in combination with fulvestrant or palbociclib in post-CDK4/6 inhibitor disease (NCT04553133) and in dual CDK4+CDK2 blockade strategy with atirmociclib (NCT05262400). BLU-222 is under investigation in the VELA trial (NCT05252416) in combination with ribociclib and fulvestrant and is showing early signals of tolerability and pharmacodynamic activity, and INX-315 (NCT05735080) and INCB123667 (NCT05238922) are in phase 1/2 trials with dedicated HR+/HER2– post-CDK4/6 inhibitor cohorts. In a phase 3 trial, the pan-CDK2/4/6 inhibitor culmerciclib in combination with fulvestrant improved PFS vs placebo plus fulvestrant in pretreated HR+/HER2– breast cancer (16.6 vs 7.5 months; HR, 0.36; 95% CI, 0.26-0.51; P<.0001), with an ORR of 40.2% vs 12.1%. These findings provide the most mature data indicating that concurrent CDK2/4/6 blockade can meaningfully overcome endocrine resistance in this setting.155
Disclosures
None of the authors have any disclosures.
References
1. American Cancer Society. Breast Cancer Facts & Figures 2024-2025. https://www.cancer.org/content/dam/cancer-org/research/cancer-facts-and-statistics/breast-cancer-facts-and-figures/2024/breast-cancer-facts-and-figures-2024.pdf
2. Giaquinto AN, Sung H, Newman LA, et al. Breast cancer statistics 2024. CA Cancer J Clin. 2024;74(6):477-495.
3. Perou CM, Sørlie T, Eisen MB, et al. Molecular portraits of human breast tumours. Nature. 2000;406(6797):747-752.
4. Parker JS, Mullins M, Cheang MCU, et al. Supervised risk predictor of breast cancer based on intrinsic subtypes. J Clin Oncol. 2009;27(8):1160-1167.
5. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA. 1977;74(12):5463-5467.
6. Watson JD, Crick FHC. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature. 1953;171(4356):737-738.
7. Kumar KR, Cowley MJ, Davis RL. Next-generation sequencing and emerging technologies. Semin Thromb Hemost. 2019;45(7):661-673.
8. Verigos J, Magklara A. Revealing the complexity of breast cancer by next generation sequencing. Cancers (Basel). 2015;7(4):2183-2200.
9. Belkadi A, Bolze A, Itan Y, et al. Whole-genome sequencing is more powerful than whole-exome sequencing for detecting exome variants. Proc Natl Acad Sci USA. 2015;112(17):5473-5478.
10. Eichler EE. Genetic variation, comparative genomics, and the diagnosis of disease. N Engl J Med. 2019;381(1):64-74.
11. Ganatra H, Tan JK, Simmons A, et al. Applying whole-genome and whole-exome sequencing in breast cancer: a review of the landscape. Breast Cancer. 2024;31(6):999-1009.
12. Ramarao-Milne P, Kondrashova O, Patch AM, et al. Comparison of actionable events detected in cancer genomes by whole-genome sequencing, in silico whole-exome and mutation panels. ESMO Open. 2022;7(4):100540.
13. Lee W-C. Whole genome sequencing for more accurate TMB and mutational signatures in common cancers [ASCO abstract e15173]. J Clin Oncol. 2023;41(suppl)(16).
14. Lionel AC, Costain G, Monfared N, et al. Improved diagnostic yield compared with targeted gene sequencing panels suggests a role for whole-genome sequencing as a first-tier genetic test. Genet Med. 2018;20(4):435-443.
15. Miller EM, Patterson NE, Zechmeister JM, et al. Development and validation of a targeted next generation DNA sequencing panel outperforming whole exome sequencing for the identification of clinically relevant genetic variants. Oncotarget. 2017;8(60):102033-102045.
16. McCabe MJ, Gauthier MA, Chan CL, et al; kConFab. Development and validation of a targeted gene sequencing panel for application to disparate cancers. Sci Rep. 2019;9(1):17052.
17. Frampton GM, Fichtenholtz A, Otto GA, et al. Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing. Nat Biotechnol. 2013;31(11):1023-1031.
18. Siravegna G, Mussolin B, Venesio T, et al. How liquid biopsies can change clinical practice in oncology. Ann Oncol. 2019;30(10):1580-1590.
19. Anitha K, Posinasetty B, Naveen Kumari K, et al. Liquid biopsy for precision diagnostics and therapeutics. Clin Chim Acta. 2024;554:117746.
20. Nikanjam M, Kato S, Kurzrock R. Liquid biopsy: current technology and clinical applications. J Hematol Oncol. 2022;15(1):131.
21. Yazaki S, Tokura M, Aiba H, Kojima Y, Shiraishi K. Clinical applications of cell-free DNA-based liquid biopsy analysis. Transl Oncol. 2025;61:102519.
22. Carr DJ, Welch HG. Assessing the clinical utility of liquid biopsies across 5 potential indications from therapy selection to population screening: a review. JAMA Intern Med. 2023;183(10):1144-1151.
23. FDA approves Foundation Medicine’s FoundationOne CDx, the first pan-tumour comprehensive genomic profiling assay incorporating a broad range of companion diagnostics [press release]. Posted December 03, 2017. Accessed August 7, 2026. https://www.roche.com/investors/updates/inv-update-2017-12-04.
24. Cocco E, Benhamida J, Middha S, et al. Tearing down the walls: FDA approves next generation sequencing (NGS) assays for actionable cancer genomic aberrations. J Exp Clin Cancer Res. 2018;37:47.
25. Milbury CA, Creeden J, Yip WK, et al. Clinical and analytical validation of FoundationOne®CDx, a comprehensive genomic profiling assay for solid tumors. PLoS One. 2022;17(3):e0264138.
26. Andre F, Filleron T, Kamal M, et al. Genomics to select treatment for patients with metastatic breast cancer. Nature. 2022;610(7931):343-348.
27. Canino F, Tornincasa A, Bettelli S, et al. Real-world data and clinical implications of next-generation sequencing (NGS)-based analysis in metastatic breast cancer patients. Int J Mol Sci. 2024;25(5):5.
28. Suh KJ, Kim SH, Kim YJ, et al. Clinical application of next-generation sequencing in patients with breast cancer: real-world data. J Breast Cancer. 2022;25(5):366-378.
29. Hempel D, Ebner F, Garg A, et al. Real world data analysis of next generation sequencing and protein expression in metastatic breast cancer patients. Sci Rep. 2020;10(1):10459.
30. Brett JO, Spring LM, Bardia A, Wander SA. ESR1 mutation as an emerging clinical biomarker in metastatic hormone receptor-positive breast cancer. Breast Cancer Res. 2021;23(1):85.
31. Wander SA, Cohen O, Gong X, et al. The genomic landscape of intrinsic and acquired resistance to cyclin-dependent kinase 4/6 inhibitors in patients with hormone receptor-positive metastatic breast cancer. Cancer Discov. 2020;10(8):1174-1193.
32. Hong D, Infante J, Janku F, et al. Phase I study of LY2606368, a checkpoint kinase 1 inhibitor, in patients with advanced cancer. J Clin Oncol. 2016;34(15):1764-1771.
33. Haddad TC, D’Assoro A, Suman V, et al. Phase I trial to evaluate the addition of alisertib to fulvestrant in women with endocrine-resistant, ER+ metastatic breast cancer. Breast Cancer Res Treat. 2018;168(3):639-647.
34. Gerosa R, De Sanctis R, Jacobs F, et al. Cyclin-dependent kinase 2 (CDK2) inhibitors and others novel CDK inhibitors (CDKi) in breast cancer: clinical trials, current impact, and future directions. Crit Rev Oncol Hematol. 2024;196:104324.
35. Marguerat S, Bähler J. RNA-seq: from technology to biology. Cell Mol Life Sci. 2010;67(4):569-579.
36. Stark R, Grzelak M, Hadfield J. RNA sequencing: the teenage years. Nat Rev Genet. 2019;20(11):631-656.
37. Smith CC, Bixby LM, Miller KL, et al. Using RNA sequencing to characterize the tumor microenvironment. Methods Mol Biol. 2020;2055:245-272.
38. Tsang ES, Grisdale CJ, Pleasance E, et al. Uncovering clinically relevant gene fusions with integrated genomic and transcriptomic profiling of metastatic cancers. Clin Cancer Res. 2021;27(2):522-531.
39. Benayed R, Offin M, Mullaney K, et al. High yield of RNA sequencing for targetable kinase fusions in lung adenocarcinomas with no mitogenic driver alteration detected by DNA sequencing and low tumor mutation burden. Clin Cancer Res. 2019;25(15):4712-4722.
40. Michuda J, Park BH, Cummingsi AL, et al. Use of clinical RNA-sequencing in the detection of actionable fusions compared to DNA-sequencing alone [ASCO abstract 3077]. J Clin Oncol. 2022;40(16)(suppl).
41. Angus L, Smid M, Wilting SM, et al; Cpct Consortium. Genomic alterations associated with estrogen receptor pathway activity in metastatic breast cancer have a differential impact on downstream ER signaling. Cancers (Basel). 2023;15(17):4416.
42. Xia Y, He X, Renshaw L, et al. Integrated DNA and RNA sequencing reveals drivers of endocrine resistance in estrogen receptor-positive breast cancer. Clin Cancer Res. 2022;28(16):3618-3629.
43. Sparano J, O’Neill A, Alpaugh K, et al. Association of circulating tumor cells with late recurrence of estrogen receptor-positive breast cancer: a secondary analysis of a randomized clinical trial. JAMA Oncol. 2018;4(12):1700-1706.
44. Ningsi R, Elazezy M, Stegat L, et al. Detection and characterization of estrogen receptor alfa expression of circulating tumor cells as a prognostic marker. Cancers (Basel). 2022;14(11):2621.
45. Fabisiewicz A, Szostakowska-Rodzos M, Zaczek AJ, Grzybowska EA. Circulating tumor cells in early and advanced breast cancer; biology and prognostic value. Int J Mol Sci. 2020;21(5):1671.
46. Mouhanna P, Ståhlberg A, Andersson D, et al. Integration of personalised ultrasensitive ctDNA monitoring of patients with metastatic breast cancer to reduce imaging requirements. Int J Cancer. 2025;156(8):1509-1517.
47. André F, Ciruelos EM, Juric D, et al. Alpelisib plus fulvestrant for PIK3CA-mutated, hormone receptor-positive, human epidermal growth factor receptor-2-negative advanced breast cancer: final overall survival results from SOLAR-1. Ann Oncol. 2021;32(2):208-217.
48. Higgins MJ, Jelovac D, Barnathan E, et al. Detection of tumor PIK3CA status in metastatic breast cancer using peripheral blood. Clin Cancer Res. 2012;18(12):3462-3469.
49. Chae YK, Davis AA, Jain S, et al. Concordance of genomic alterations by next-generation sequencing in tumor tissue versus circulating tumor DNA in breast cancer. Mol Cancer Ther. 2017;16(7):1412-1420.
50. Davis AA, Jacob S, Gerratana L, et al. Landscape of circulating tumour DNA in metastatic breast cancer. EBioMedicine. 2020;58:102914.
51. Tolaney SM, Toi M, Neven P, et al. Clinical significance of PIK3CA and ESR1 mutations in circulating tumor DNA: analysis from the MONARCH 2 study of abemaciclib plus fulvestrant. Clin Cancer Res. 2022;28(8):1500-1506.
52. Venetis K, Pepe F, Pescia C, et al. ESR1 mutations in HR+/HER2-metastatic breast cancer: enhancing the accuracy of ctDNA testing. Cancer Treat Rev. 2023;121:102642.
53. Bidard FC, Mayer EL, Park YH, et al; SERENA-6 Study Group. First-line camizestrant for emerging ESR1-mutated advanced breast cancer. N Engl J Med. 2025;393(6):569-580.
54. Mögele T, Hildebrand K, Sultan A, et al. Dissecting tumor heterogeneity by liquid biopsy-a comparative analysis of post-mortem tissue and pre-mortem liquid biopsies in solid neoplasias. Int J Mol Sci. 2025;26(15):7614.
55. Gristina V, Pepe F, Ogliari FR, et al. Smaller, cheaper, faster: where next for liquid biopsies? Expert Rev Mol Diagn. 2025;25(9):555-573.
56. Hofman P. Next-generation sequencing with liquid biopsies from treatment-naïve non-small cell lung carcinoma patients. Cancers (Basel). 2021;13(9):2049.
57. Chen M, Zhao H. Next-generation sequencing in liquid biopsy: cancer screening and early detection. Hum Genomics. 2019;13(1):34.
58. Heitzer E, Haque IS, Roberts CES, Speicher MR. Current and future perspectives of liquid biopsies in genomics-driven oncology. Nat Rev Genet. 2019;20(2):71-88.
59. Lin LH, Allison DHR, Feng Y, et al. Comparison of solid tissue sequencing and liquid biopsy accuracy in identification of clinically relevant gene mutations and rearrangements in lung adenocarcinomas. Mod Pathol. 2021;34(12):2168-2174.
60. Lin Z, Li Y, Tang S, Deng Q, Jiang J, Zhou C. Comparative analysis of genomic profiles between tissue-based and plasma-based next-generation sequencing in patients with non-small cell lung cancer. Lung Cancer. 2023;182:107282.
61. Esagian SM, Grigoriadou GΙ, Nikas IP, et al. Comparison of liquid-based to tissue-based biopsy analysis by targeted next generation sequencing in advanced non-small cell lung cancer: a comprehensive systematic review. J Cancer Res Clin Oncol. 2020;146(8):2051-2066.
62. Mahuron KM, Fong Y. Applications of liquid biopsy for surgical patients with cancer: a review. JAMA Surg. 2024;159(1):96-103.
63. Meintani A, Ozdogan M, Touroutoglou N, et al. Comprehensive evaluation of a 1021-gene panel in FFPE and liquid biopsy for analytical and clinical use. Int J Mol Sci. 2025;26(13):5930.
64. FoundationOne CDx. Foundation Medicine. Accessed July 20, 2026. https://www.foundationmedicine.com/test/foundationone-cdx
65. MSK-IMPACT: a comprehensive tumor sequencing test to detect targetable DNA mutations. Memorial Sloan Kettering Cancer Center. Accessed July 20, 2026. https://www.mskcc.org/departments/division-solid-tumor-oncology/early-drug-development-service-phase-clinical-trials/precision-medicine-approach/msk-impact
66. Cheng DT, Mitchell TN, Zehir A, et al. Memorial Sloan Kettering-integrated mutation profiling of actionable cancer targets (MSK-IMPACT): a hybridization capture-based next-generation sequencing clinical assay for solid tumor molecular oncology. J Mol Diagn. 2015;17(3):251-264.
67. MSK-ACCESS: A liquid biopsy test that offers noninvasive cancer genomic profiling. Memorial Sloan Kettering Cancer Center. Accessed July 20, 2026. https://www.mskcc.org/msk-access
68. OmniSeq INSIGHT, Solid Tumor NGS Panel (DNA and RNA). Labcorp Oncology. Accessed July 20, 2026. https://oncology.labcorp.com/tests/831177/omniseq-insight-solid-tumor-ngs-panel-dna-and-rna
69. Guardant Complete: testing across the continuum of cancer. Guardant Health. Accessed July 20, 2026. https://guardanthealth.com/products/tests-for-patients-with-early-and-advanced-stage-cancer/
70. Abbasi HQ, Maryyum A, Khan AM, Shahnoor S, Oduoye MO, Wechuli PN. Advancing precision oncology in breast cancer: the FDA approval of elacestrant and Guardant360 CDx: a correspondence. Int J Surg. 2023;109(7):2157-2158.
71. Guardant360 Liquid CDx. Guardant Health. Accessed July 20, 2026. https://www.guardantcomplete.com/hcp/solutions/guardant360-liquid
72. Nuzhdina K, Lozinsky Y, Podsvirova S, et al. Analytical and clinical validation of the BostonGene tumor portrait assay [ASCO abstract e15085]. J Clin Oncol. 2021;39(15)(suppl).
73. Providers. BostonGene. Accessed July 20, 2026. https://www.bostongene.com/providers
74. Beaubier N, Tell R, Lau D, et al. Clinical validation of the tempus xT next-generation targeted oncology sequencing assay. Oncotarget. 2019;10(24):2384-2396.
75. Tempus xT CDx FDA-approved molecular profiling for solid tumors. Tempus. Accessed August 6, 2026. https://www.tempus.com/solutions/xt-cdx/
76.Tempus xF/xF+ liquid biopsy. Tempus. Accessed July 20, 2026. https://www.tempus.com/oncology/genomic-profiling/xf/
77. Premarket Approval (PMA). US Food & Drug Administration. Accessed July 20, 2026. https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpma/pma.cfm?id=P240010
78. MI cancer seek. Caris Life Sciences. Accessed July 20, 2026. https://www.carislifesciences.com/physicians/physician-tests/mi-cancer-seek/
79. Caris Assure. Caris Life Sciences. Accessed July 20, 2026. https://www.carislifesciences.com/physicians/physician-tests/caris-assure/
80. Oncomine Breast cfDNA Assay. Accessed July 20, 2026. https://www.thermofisher.com/order/catalog/product/A31183
81. Page K, Martinson LJ, Fernandez-Garcia D, et al. Circulating tumor DNA profiling from breast cancer screening through to metastatic disease [published online November 24, 2021]. JCO Precis Oncol.
82. Conroy JM, Pabla S, Glenn ST, et al. A scalable high-throughput targeted next-generation sequencing assay for comprehensive genomic profiling of solid tumors [published online December 2, 2021]. PLoS One. doi:10.1371/journal.pone.0260089.
83. TruSight Oncology 500 High-Throughput. Illumina. Accessed July 20, 2026. https://www.illumina.com/products/by-type/clinical-research-products/trusight-oncology-500-ht.html
84. TruSight Oncology 500 ctDNA v2. Illumina. Accessed July 20, 2026. https://www.illumina.com/products/by-type/clinical-research-products/trusight-oncology-500-ctdna.html
85. Al Sukhun S, Temin S, Barrios CH, et al. Systemic treatment of patients with metastatic breast cancer: ASCO resource-stratified guideline. JCO Glob Oncol. 2024;10(10):e2300285.
86. National Comprehensive Cancer Network (NCCN) clinical guidelines: invasive breast cancer. Version 5.2026. July 7, 2026. Accessed July 20, 2026. https://www.nccn.org/professionals/physician_gls/pdf/breast.pdf
87. McAndrew NP, Finn RS. Clinical review on the management of hormone receptor-positive metastatic breast cancer. JCO Oncol Pract. 2022;18(5):319-327.
88. D’Amico P, Cristofanilli M. Standard of care in hormone receptor-positive metastatic breast cancer: can we improve the current regimens or develop better selection tools? JCO Oncol Pract. 2022;18(5):331-334.
89. Turner NC, Slamon DJ, Ro J, et al. Overall survival with palbociclib and fulvestrant in advanced breast cancer. N Engl J Med. 2018;379(20):1926-1936.
90. Slamon DJ, Neven P, Chia S, et al. Overall survival with ribociclib plus fulvestrant in advanced breast cancer. N Engl J Med. 2020;382(6):514-524.
91. Sledge GW Jr, Toi M, Neven P, et al. The effect of abemaciclib plus fulvestrant on overall survival in hormone receptor-positive, ERBB2-negative breast cancer that progressed on endocrine therapy-MONARCH 2: a randomized clinical trial. JAMA Oncol. 2020;6(1):116-124.
92. Goetz MP, Toi M, Campone M, et al. MONARCH 3: abemaciclib as initial therapy for advanced breast cancer. J Clin Oncol. 2017;35(32):3638-3646.
93. Finn RS, Martin M, Rugo HS, et al. Palbociclib and letrozole in advanced breast cancer. N Engl J Med. 2016;375(20):1925-1936.
94. Hortobagyi GN, Stemmer SM, Burris HA, et al. Ribociclib as first-line therapy for HR-positive, advanced breast cancer. N Engl J Med. 2016;375(18):1738-1748.
95. Cardoso F, Costa A, Senkus E, et al. 3rd ESO-ESMO international consensus guidelines for advanced breast cancer (ABC 3). Ann Oncol. 2017;28(1):16-33.
96. D’Souza A, Spicer D, Lu J. Overcoming endocrine resistance in metastatic hormone receptor-positive breast cancer. J Hematol Oncol. 2018;11(1):80.
97. Yang SX, Polley E, Lipkowitz S. New insights on PI3K/AKT pathway alterations and clinical outcomes in breast cancer. Cancer Treat Rev. 2016;45:87-96.
98. Chen JW, Murugesan K, Newberg JY, et al. Comparison of PIK3CA mutation prevalence in breast cancer across predicted ancestry populations. JCO Precis Oncol. 2022;6(6):e2200341.
99. Turner NC, Oliveira M, Howell SJ, et al; CAPItello-291 Study Group. Capivasertib in hormone receptor-positive advanced breast cancer. N Engl J Med. 2023;388(22):2058-2070.
100. Millis SZ, Ikeda S, Reddy S, Gatalica Z, Kurzrock R. Landscape of phosphatidylinositol-3-kinase pathway alterations across 19 784 diverse solid tumors. JAMA Oncol. 2016;2(12):1565-1573.
101. Rugo HS, Lerebours F, Ciruelos E, et al. Alpelisib plus fulvestrant in PIK3CA-mutated, hormone receptor-positive advanced breast cancer after a CDK4/6 inhibitor (BYLieve): one cohort of a phase 2, multicentre, open-label, non-comparative study. Lancet Oncol. 2021;22(4):489-498.
102. Howell SJ, Casbard A, Carucci M, et al. Fulvestrant plus capivasertib versus placebo after relapse or progression on an aromatase inhibitor in metastatic, oestrogen receptor-positive, HER2-negative breast cancer (FAKTION): overall survival, updated progression-free survival, and expanded biomarker analysis from a randomised, phase 2 trial. Lancet Oncol. 2022;23(7):851-864.
103. Turner NC, Im SA, Saura C, et al. Inavolisib-based therapy in PIK3CA-mutated advanced breast cancer. N Engl J Med. 2024;391(17):1584-1596.
104. Jhaveri KL, Im SA, Saura C, et al. Overall survival with inavolisib in PIK3CA-mutated advanced breast cancer. N Engl J Med. 2025;393(2):151-161.
105. Hurvitz SA, Layman RM, Curigliano G, et al; VIKTORIA-1 Study Group. VIKTORIA-1 trial of gedatolisib plus fulvestrant with or without palbociclib in hormone receptor-positive/HER2-/PIK3CA wild-type advanced breast cancer. J Clin Oncol. 2026;44(12):1108-1119.
106. Bachelot T, Bourgier C, Cropet C, et al. Randomized phase II trial of everolimus in combination with tamoxifen in patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative metastatic breast cancer with prior exposure to aromatase inhibitors: a GINECO study. J Clin Oncol. 2012;30(22):2718-2724.
107. Yardley DA, Noguchi S, Pritchard KI, et al. Everolimus plus exemestane in postmenopausal patients with HR(+) breast cancer: BOLERO-2 final progression-free survival analysis. Adv Ther. 2013;30(10):870-884.
108. Kornblum N, Zhao F, Manola J, et al. Randomized phase II trial of fulvestrant plus everolimus or placebo in postmenopausal women with hormone receptor-positive, human epidermal growth factor receptor 2-negative metastatic breast cancer resistant to aromatase inhibitor therapy: results of PrE0102. J Clin Oncol. 2018;36(16):1556-1563.
109. Vasseur A, Cabel L, Hego C, et al. Fulvestrant and everolimus efficacy after CDK4/6 inhibitor: a prospective study with circulating tumor DNA analysis. Oncogene. 2024;43(16):1214-1222.
110. Schmid P, Zaiss M, Harper-Wynne C, et al. Fulvestrant plus vistusertib vs fulvestrant plus everolimus vs fulvestrant alone for women with hormone receptor-positive metastatic breast cancer: the MANTA phase 2 randomized clinical trial. JAMA Oncol. 2019;5(11):1556-1564.
111. Toy W, Shen Y, Won H, et al. ESR1 ligand-binding domain mutations in hormone-resistant breast cancer. Nat Genet. 2013;45(12):1439-1445.
112. Burstein HJ, DeMichele A, Somerfield MR, Henry NL; Biomarker Testing and Endocrine and Targeted Therapy in Metastatic Breast Cancer Expert Panels. Testing for ESR1 mutations to guide therapy for hormone receptor-positive, human epidermal growth factor receptor 2-negative metastatic breast cancer: ASCO guideline rapid recommendation update. J Clin Oncol. 2023;41(18):3423-3425.
113. Zundelevich A, Dadiani M, Kahana-Edwin S, et al. ESR1 mutations are frequent in newly diagnosed metastatic and loco-regional recurrence of endocrine-treated breast cancer and carry worse prognosis. Breast Cancer Res. 2020;22(1):16.
114. Jeselsohn R, Yelensky R, Buchwalter G, et al. Emergence of constitutively active estrogen receptor-α mutations in pretreated advanced estrogen receptor-positive breast cancer. Clin Cancer Res. 2014;20(7):1757-1767.
115. Reinert T, Saad ED, Barrios CH, Bines J. Clinical implications of ESR1 mutations in hormone receptor-positive advanced breast cancer. Front Oncol. 2017;7:26.
116. Herzog SK, Fuqua SAW. ESR1 mutations and therapeutic resistance in metastatic breast cancer: progress and remaining challenges. Br J Cancer. 2022;126(2):174-186.
117. Bidard FC, Kaklamani VG, Neven P, et al. Elacestrant (oral selective estrogen receptor degrader) versus standard endocrine therapy for estrogen receptor-positive, human epidermal growth factor receptor 2-negative advanced breast cancer: results from the randomized phase III EMERALD trial. J Clin Oncol. 2022;40(28):3246-3256.
118. Bardia A, Cortés J, Bidard FC, et al. Elacestrant in ER+, HER2- metastatic breast cancer with ESR1-mutated tumors: subgroup analyses from the phase III EMERALD trial by prior duration of endocrine therapy plus CDK4/6 inhibitor and in clinical subgroups. Clin Cancer Res. 2024;30(19):4299-4309.
119. Rugo H, Tolaney SM, Chan N, et al. Elacestrant in combination with everolimus or abemaciclib in patients with ER+/HER2- locally advanced or metastatic breast cancer (mBC): phase 2 results from ELEVATE, an open-label, umbrella study [SABCS abstract RF7-01]. Clin Cancer Res. 2026;32(4)(suppl).
120. Bidard FC, Hardy-Bessard AC, Dalenc F, et al; PADA-1 investigators. Switch to fulvestrant and palbociclib versus no switch in advanced breast cancer with rising ESR1 mutation during aromatase inhibitor and palbociclib therapy (PADA-1): a randomised, open-label, multicentre, phase 3 trial. Lancet Oncol. 2022;23(11):1367-1377.
121. Jhaveri KL, Neven P, Casalnuovo ML, et al; EMBER-3 Study Group. Imlunestrant with or without abemaciclib in advanced breast cancer. N Engl J Med. 2025;392(12):1189-1202.
122. Jhaveri KL, Neven P, Casalnuovo ML, et al. Imlunestrant with or without abemaciclib in advanced breast cancer: updated efficacy results from the phase III EMBER-3 trial. Ann Oncol. 2026;37(4):532-543.
123. Mayer EL, Tolaney SM, Brufsky A, et al. EvERA breast cancer (BC): phase III study of giredestrant + everolimus vs exemestane + everolimus in patients (pts) with estrogen receptor-positive, HER2-negative locally advanced or metastatic BC (ER+, HER2– LA/mBC) [ASCO abstract TPS1119]. J Clin Oncol. 2023;41(16)(suppl).
124. Rugo HS, Tolaney SM, Jhaveri KL, et al. Clinical and biomarker subgroup analysis from the phase 3 evERA trial: giredestrant plus everolimus in ER-positive/HER2-negative advanced breast cancer after CDK4/6 inhibitor treatment [SABCS abstract GS3-09]. Clin Cancer Res. 2026;32(4)(suppl).
125. Malone KE, Daling JR, Doody DR, et al. Prevalence and predictors of BRCA1 and BRCA2 mutations in a population-based study of breast cancer in white and black American women ages 35 to 64 years. Cancer Res. 2006;66(16):8297-8308.
126. Robson M, Im SA, Senkus E, et al. Olaparib for metastatic breast cancer in patients with a germline BRCA mutation. N Engl J Med. 2017;377(6):523-533.
127. Robson ME, Tung N, Conte P, et al. OlympiAD final overall survival and tolerability results: olaparib versus chemotherapy treatment of physician’s choice in patients with a germline BRCA mutation and HER2-negative metastatic breast cancer. Ann Oncol. 2019;30(4):558-566.
128. Litton JK, Rugo HS, Ettl J, et al. Talazoparib in patients with advanced breast cancer and a germline BRCA mutation. N Engl J Med. 2018;379(8):753-763.
129. Litton JK, Hurvitz SA, Mina LA, et al. Talazoparib versus chemotherapy in patients with germline BRCA1/2-mutated HER2-negative advanced breast cancer: final overall survival results from the EMBRACA trial. Ann Oncol. 2020;31(11):1526-1535.
130. Bruno L, Ostinelli A, Waisberg F, et al. Cyclin-dependent kinase 4/6 inhibitor outcomes in patients with advanced breast cancer carrying germline pathogenic variants in DNA repair-related genes. JCO Precis Oncol. 2022;6:e2100140.
131. Berton Giachetti PPM, Morganti S, Gandini S, et al. Survival following CDK4/6 inhibitor therapy for hormone receptor-positive, ERBB2-negative metastatic breast cancer. JAMA Netw Open. 2025;8(2):e2461067.
132. Tung NM, Robson ME, Ventz S, et al. TBCRC 048: phase II study of olaparib for metastatic breast cancer and mutations in homologous recombination-related genes. J Clin Oncol. 2020;38(36):4274-4282.
133. Tung NM, Robson ME, Nanda R, et al. TBCRC 048 (olaparib expanded) expansion cohorts: phase 2 study of olaparib monotherapy in patients (pts) with metastatic breast cancer (MBC) with germline (g) mutations in PALB2 or somatic (s) mutations in BRCA1 or BRCA2 [ASCO abstract 1021]. J Clin Oncol. 2024;42(16)(suppl).
134. Schlam I, Tolaney SM, Tarantino P. How I treat HER2-low advanced breast cancer. Breast. 2023;67:116-123.
135. Schettini F, Chic N, Brasó-Maristany F, et al. Clinical, pathological, and PAM50 gene expression features of HER2-low breast cancer. NPJ Breast Cancer. 2021;7(1):1.
136. Modi S, Jacot W, Yamashita T, et al; DESTINY-Breast04 Trial Investigators. Trastuzumab deruxtecan in previously treated HER2-low advanced breast cancer. N Engl J Med. 2022;387(1):9-20.
137. Bardia A, Hu X, Dent R, et al; DESTINY-Breast06 Trial Investigators. Trastuzumab deruxtecan after endocrine therapy in metastatic breast cancer. N Engl J Med. 2024;391(22):2110-2122.
138. FDA approves fam-trastuzumab deruxtecan-nxki for unresectable or metastatic HR-positive, HER2-low or HER2-ultralow breast cancer. US Food and Drug Administration. Accessed July 20, 2026. https://www.fda.gov/drugs/resources-information-approved-drugs/fda-approves-fam-trastuzumab-deruxtecan-nxki-unresectable-or-metastatic-hr-positive-her2-low-or-her2
139. Ma CX, Luo J, Freedman RA, et al. The phase II MutHER study of neratinib alone and in combination with fulvestrant in HER2-mutated, non-amplified metastatic breast cancer. Clin Cancer Res. 2022;28(7):1258-1267.
140. Lien HC, Chen YL, Juang YL, Jeng YM. Frequent alterations of HER2 through mutation, amplification, or overexpression in pleomorphic lobular carcinoma of the breast. Breast Cancer Res Treat. 2015;150(2):447-455.
141. Jhaveri K, Eli LD, Wildiers H, et al. Neratinib + fulvestrant + trastuzumab for HR-positive, HER2-negative, HER2-mutant metastatic breast cancer: outcomes and biomarker analysis from the SUMMIT trial. Ann Oncol. 2023;34(10):885-898.
142. Okines AFC, Curigliano G, Mizuno N, et al. Tucatinib and trastuzumab in HER2-mutated metastatic breast cancer: a phase 2 basket trial. Nat Med. 2025;31(3):909-916.
143. Christofyllakis K, Bittenbring JT, Thurner L, et al. Cost-effectiveness of precision cancer medicine-current challenges in the use of next generation sequencing for comprehensive tumour genomic profiling and the role of clinical utility frameworks (review). Mol Clin Oncol. 2022;16(1):21.
144. Papalexis P, Georgakopoulou VE, Drossos PV, Thymara E, Nonni A, Lazaris AC, Zografos GC, Spandidos DA, Kavantzas N, Thomopoulou GE. Precision medicine in breast cancer (review). Mol Clin Oncol. 2024;21(5):78.
145. Bouchal P, Schubert OT, Faktor J, et al. Breast cancer classification based on proteotypes obtained by SWATH mass spectrometry. Cell Rep. 2019;28(3):832-843.e7.
146. Beatty A, Fink LS, Singh T, et al. Metabolite profiling reveals the glutathione biosynthetic pathway as a therapeutic target in triple-negative breast cancer. Mol Cancer Ther. 2018;17(1):264-275.
147. Sebastian W, Forchette L, Donoughe K, Lun Y, Verma A, Liu T. Genetics, treatment, and new technologies of hormone receptor-positive breast cancer. Cancers (Basel). 2023;15(4):4.
148. Pourtau L, Oliveira H, Thevenot J, et al. Antibody-functionalized magnetic polymersomes: in vivo targeting and imaging of bone metastases using high resolution MRI. Adv Healthc Mater. 2013;2(11):1420-1424.
149. Guo P, Yang J, Huang J, Auguste DT, Moses MA. Therapeutic genome editing of triple-negative breast tumors using a noncationic and deformable nanolipogel. Proc Natl Acad Sci USA. 2019;116(37):18295-18303.
150. Paoletti L, Zoratto N, Benvenuto M, et al. Hyaluronan-estradiol nanogels as potential drug carriers to target ER+ breast cancer cell line. Carbohydr Polym. 2023;314:120900.
151. Li B, Qi F, Zhu F, et al. Nanoparticle-based combination therapy enhances fulvestrant efficacy and overcomes tumor resistance in ER-positive breast cancer. Cancer Res. 2023;83(17):2924-2937.
152. A phase 1/2 trial of ARV-471 alone and in combination with palbociclib (IBRANCE®) in patients with ER+/HER2- locally advanced or metastatic breast cancer (mBC). Accessed July 20, 2026. https://clinicaltrials.gov/study/NCT04072952
153. Campone M, De Laurentiis M, Jhaveri K, et al; VERITAC-2 Study Group. Vepdegestrant, a PROTAC estrogen receptor degrader, in advanced breast cancer. N Engl J Med. 2025;393(6):556-568.
154. Tzetzo SL, Schultz E, Wang J, et al. Baseline cell cycle and immune profiles indicate CDK4/6 inhibitor response in metastatic HR + /HER2- breast cancer. NPJ Breast Cancer. 2025;11(1):54.
155. Yin Y, Zhang Q, Sun T, et al. Novel CDK2/4/6 inhibitor culmerciclib (TQB3616) plus fulvestrant in previously treated, HR-positive, HER2-negative advanced breast cancer: a randomized, double-blind, phase 3 trial. Signal Transduct Target Ther. 2025;10(1):414.