Point mutations in the ligand binding domain of retinoic acid receptor alpha (RARα) are linked to breast fibroepithelial tumor development, but their role in solid tumorigenesis is unclear. In this study, we assessed the functional effects of known RARα mutations on retinoic acid signaling using biochemical and cellular assays. All tested mutants exhibited reduced transcriptional activity compared to wild-type RARα and showed a dominant negative effect, a feature associated with developmental defects and tumor formation. X-ray crystallography revealed that the mutants maintained structural integrity, with altered co-activator recruitment explaining the loss of transcriptional function. Transcriptomics and cell growth assays demonstrated that mutant RARα proteins conferred resistance to ligand-induced growth inhibition in phyllodes tumor cells. Although the mutations impair RARα's response to retinoic acid, some mutants could be partially reactivated with synthetic agonists. These findings provide insights into how RARα mutations may contribute to tumorigenesis.

Endocrine therapy with CDK4/6 inhibitors is standard for estrogen receptor-positive, HER2-negative metastatic breast cancer (ER+/HER2- MBC), yet clinical resistance develops. Previously, we demonstrated that low doses of palbociclib activate autophagy, reversing initial G1 cell cycle arrest, while high concentrations induce off-target senescence. The autophagy inhibitor hydroxychloroquine (HCQ) induced on-target senescence at lower palbociclib doses. We conducted a phase I trial (NCT03774472 registered in ClinicalTrials.gov on 8/20/2018) of HCQ (400, 600, 800 mg/day) with palbociclib (75 mg/day continuous) and letrozole, using a 3 + 3 design. Primary objectives included safety, tolerability, and determining the recommended phase 2 dose (RP2D) of HCQ. Secondary objectives included tumor response and biomarker analysis. Fourteen ER+/HER2- MBC patients were evaluable [400 mg (n = 4), 600 mg (n = 4), 800 mg (n = 6)]. Grade 3 adverse events (AEs) included hematological (3 at 800 mg), skin rash (2 at 600 mg), and anorexia (1 at 400 mg), with no serious AEs. The best responses were partial (2), stable (11), and progression (1). Tumor reductions ranged from 11% to 30%, with one 55% increase. The two partial responders sustained tumor size reductions of 30% to 55% over an extended treatment period, lasting nearly 300 days. Biomarker analysis in responders demonstrated significant decreases in Ki67, Rb, and nuclear cyclin E levels and increases in autophagy markers p62 and LAMP1, suggesting a correlation between these biomarkers and treatment response. This phase I study demonstrated that HCQ is safe and well-tolerated and the RP2D was established at 800 mg/day with continuous low-dose palbociclib (75 mg/day) and letrozole (2.5 mg/day). These findings suggest that adding HCQ could potentially enhance the efficacy of low-dose palbociclib and standard letrozole therapy, pending verification in larger randomized studies.

Patients with metastatic breast cancer face reduced quality of life and increased mortality rates, necessitating more effective anti-cancer strategies. Building on previous research that identified metastatic-niche-specific metabolic vulnerabilities, we investigated how a ketogenic diet enhances estrogen receptor (ER)-positive liver metastatic breast cancer's response to Fulvestrant (Fulv) treatment. Using in vitro cell lines and in vivo xenograft metastasis mouse models, we examined the molecular mechanisms of combining ER targeting with a ketogenic diet. We found that Fulv treatment downregulates the ketogenesis pathway enzyme OXCT1, leading to β-hydroxybutyrate accumulation and decreased tumor cell viability. We also explored interactions between glucose, palmitic acid, and β-hydroxybutyric acid. These findings establish the molecular basis and clinical potential of a ketogenic diet to enhance Fulv efficacy in patients with ER+ liver metastatic breast cancer, potentially improving survival outcomes and quality of life in this population.

Optimal timing and dosing of adjuvant cyclin-dependent kinase (CDK) 4/6 inhibitor in early breast cancer is controversial. This prospective phase II clinical trial investigated tolerability and safety of two ribociclib dosing schedules. Patients with stage I-III hormone receptor-positive (HR+)/HER2- breast cancer on adjuvant endocrine therapy (ET) were randomized to two ribociclib dosing schedules: 400 mg continuous vs 600 mg intermittent, with initiation in early (prior ET < 2 years) vs delayed (prior ET ≥ 2 years) setting. Primary objective was to evaluate safety and tolerability of continuous vs intermittent schedule. Primary endpoint was proportion of patients who discontinued ribociclib before completion of all 12 cycles (measured at 12 months). Recurrence free survival (RFS) and circulating tumor DNA (ctDNA) detection were also evaluated. 81 patients were enrolled. Only six serious adverse events occurred, with no significant difference between treatment arms and no subject deaths. Twenty-five patients (31%) discontinued ribociclib before completion of 12 months, with no significant difference between treatment arms. Ribociclib discontinuation was higher in early vs delayed initiation (36% vs 21%). At median follow-up of 20 months, two patients in the intermittent arm (600 mg; Arm 2) experienced disease recurrence (2-year RFS 97%, 95%CI 88-99%), vs none in the continuous arm (400 mg; Arm 1) (2-year RFS 100%). ctDNA was only identified in the two subjects with recurrent disease at median of 7.5 months prior to radiological recurrence. Ribociclib is a safe and well-tolerated adjunct to adjuvant ET in early-stage breast cancer. Delayed initiation of ribociclib at 400 mg continuous dosing was feasible, better tolerated and associated with promising outcomes. ctDNA detection preceded clinical evidence of recurrence and may be considered as a surveillance tool in breast cancer.

Discrimination can contribute to worse health outcomes, but its prevalence in breast cancer is not well studied. We aimed to understand how women with stage I-III breast cancer faced discrimination in health care and everyday settings through the Everyday Discrimination Scale, cross-sectional survey. 296 women, 178 (60%) Non-Hispanic White (NHW), 76 (26%) Non-Hispanic Black (NHB), and 42 (14%) Hispanic participated. NHB women reported significantly more discrimination in everyday life compared to NHW women (score 20.1 vs 16.1, p < 0.001) and Hispanic women (score 20.1 vs 16.0, p < 0.001). In the health care setting, NHB had statistically more frequent reports of being ignored (23.7% vs. 5.6%), treated with less respect (21.1% vs. 7.3%), and treated with less courtesy (18.7% vs. 6.2%; all P = < 0.001) when compared to NHW women. NHB women experience a higher degree of discrimination both inside and outside of health care. Further research to understand discrimination on breast cancer outcomes is warranted.

Cancer disrupts intratumoral innate-adaptive immune crosstalk, but how the systemic immune landscape evolves during breast cancer progression remains unclear. We profiled circulating immune cells in stage I-III and stage IV triple-negative breast cancer (TNBC) patients and healthy donors (HDs). Metastatic TNBC (mTNBC) patients had reduced T cells, dendritic cells, and differentiated B cells compared to non-metastatic TNBC patients and HDs, partly linked to prior chemotherapy. Vδ1 γδ T cells from mTNBC patients produced more IL17 than those from HDs. Chemotherapy-naïve mTNBC patients showed increased classical monocytes and neutrophils. Transcriptional, proteomic, and functional analyses revealed that neutrophils in mTNBC exhibited enhanced migratory capacity, elevated granule proteins, and higher ROS production. Some immune changes, such as reduced non-switched B cells and heightened neutrophil migration, were evident in earlier TNBC stages. This study comprehensively maps systemic immunity in TNBC, guiding future research on patient stratification and immunomodulation strategies.

Special histologic subtypes of breast cancer (BC) exhibit unique phenotypes and molecular profiles with diagnostic and therapeutic implications, often differing in behavior and clinical trajectory from common BC forms. Novel methodologies, such as artificial intelligence may improve classification. Genetic predisposition plays roles in a subset of cases. Uncommon BC presentations like male, inflammatory and pregnancy-related BC pose challenges. Emerging therapeutic strategies targeting genetic alterations or immune microenvironment are being explored.

Using a novel unsupervised method to integrate multi-omic data, we previously identified a breast cancer group with a poor prognosis. In the current study, we characterize the biological features of this subgroup, defined as the high-risk group, using various data sources. Assessment of three published hypoxia signatures showed that the high-risk group exhibited higher hypoxia scores (p < 0.0001 in all three signatures), compared to the low-risk group. Our analysis of the immune cell composition using CIBERSORT and leukocyte fraction showed significant differences between the high and low-risk groups across the entire cohort, as well as within PAM50 subtypes. Within the basal subtype, the low-risk group had a statistically significantly higher spatial fraction of tumor-infiltrating lymphocytes (TILs) compared to the high-risk group (p = 0.0362). Our findings indicate that this subgroup with poor prognosis is driven by a distinct biological signature with high activation of hypoxia-related genes as well as a low number of TILs.

Organoid cultures offer a powerful technology to investigate many different aspects of development, physiology, and pathology of diverse tissues. Unlike standard tissue culture of primary breast epithelial cells, breast organoids preserve the epithelial lineages and architecture of the normal tissue. However, existing organoid culture methods are tedious, difficult to scale, and do not robustly retain estrogen receptor (ER) expression and responsiveness in long-term culture. Here, we describe a modified culture method to generate and maintain organoids as suspension cultures in reconstituted basement membrane (™Matrigel). This method improves organoid growth and uniformity compared to the conventional Matrigel dome embedding method, while maintaining the fidelity of the three major epithelial lineages. Using this adopted method, we are able to culture and passage purified hormone sensing (HS) cells that retain ER responsiveness upon estrogen stimulation in long-term culture. This culture system presents a valuable platform to study the events involved in initiation and evolution of ER-positive breast cancer.

Endocrine therapies targeting the estrogen receptor (ER/ESR1) are the cornerstone to treat ER-positive breast cancers patients, but resistance often limits their effectiveness. Notable progress has been made although the fragmented way data is reported has reduced their potential impact. Here, we introduce EstroGene2.0, an expanded database of its precursor 1.0 version. EstroGene2.0 focusses on response and resistance to endocrine therapies in breast cancer models. Incorporating multi-omic profiling of 361 experiments from 212 studies across 28 cell lines, a user-friendly browser offers comprehensive data visualization and metadata mining capabilities ( https://estrogeneii.web.app/ ). Taking advantage of the harmonized data collection, our follow-up meta-analysis revealed transcriptomic landscape and substantial diversity in response to different classes of ER modulators. Endocrine-resistant models exhibit a spectrum of transcriptomic alterations including a contra-directional shift in ER and interferon signalings, which is recapitulated clinically. Dissecting multiple ESR1-mutant cell models revealed the different clinical relevance of cell model engineering and identified high-confidence mutant-ER targets, such as NPY1R. These examples demonstrate how EstroGene2.0 helps investigate breast cancer's response to endocrine therapies and explore resistance mechanisms.

Additional therapies are needed to improve outcomes in patients with hormone receptor-positive/human epidermal growth factor receptor 2-negative breast cancer. Research on the potential role of immunotherapy, particularly programmed cell death protein 1/programmed cell death ligand 1 inhibitors, is rapidly expanding in both the early and metastatic settings with some preliminary evidence suggesting benefit when used as part of combination therapy. Several ongoing phase 3 studies should help define their future role in treating these patients.

This study aimed to identify the clinico-pathological variables predictive of radiologic complete response (rCR) to first-line anti-HER2 therapy in patients with HER2-positive metastatic breast cancer. Patients were selected from the database of the GIM14 study and classified according to the best radiologic response obtained to first-line anti-HER2 therapy and upon time-to-treatment-discontinuation (TTD). A total of 545 patients were included in the analysis. Eighty patients experienced a rCR to first-line anti-HER2 therapy with a TTD > 3 months and HER2 Immunohistochemistry score 3+, presence of non-visceral metastases and 1 metastatic site were significantly associated with higher odds of obtaining a CR. Of the 80 patients achieving a rCR, 56 experienced a CR with a TTD > 18 months, with anti-HER2 therapy being the only variable significantly associated with a higher probability of achieving such sustained CR.

Surgery remains the primary treatment modality in the management of early-stage invasive breast cancer. Artificial intelligence (AI)-powered visualization platforms offer the compelling potential to aid surgeons in evaluating the tumor's location and morphology within the breast and accordingly optimize their surgical approach. We sought to validate an AI platform that employs dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to render three-dimensional (3D) representations of the tumor and 5 additional chest tissues, offering clear visualizations as well as functionalities for quantifying tumor morphology, tumor-to-landmark structure distances, excision volumes, and approximate surgical margins. This retrospective study assessed the visualization platform's performance on 100 cases with ground-truth labels vetted by 2 breast-specialized radiologists. We assessed features including automatic AI-generated clinical metrics (e.g., tumor dimensions) as well as visualization tools including convex hulls at desired margins around the tumor to help visualize lumpectomy volume. The statistical performance of the platform's automated features was robust and within the range of inter-radiologist variability. These detailed 3D tumor and surrounding multi-tissue depictions offer both qualitative and quantitative comprehension of cancer topology and may aid in formulating an optimal surgical approach for breast cancer treatment. We further establish the framework for broader data integration into the platform to enhance precision cancer care.

Small, hormone receptor-positive (HR+), HER2-negative (HER2-), lymph node-negative breast cancers are associated with relatively low rates of disease recurrence and have therefore been underrepresented in clinical trials assessing the effects of systemic therapy. Consequently, it remains uncertain if this patient population derives benefit from these treatments. For this exploratory analysis, we selected MINDACT (NCT00433589) patients with a HR+, HER2-, T1ab (≤1 cm) tumor and negative lymph nodes. Patients with discordant clinical risk and MammaPrint genomic risk classification were randmomized to receive chemotherapy based on either the clinical or the genomic risk assessment. Endocrine therapy treatment was based on local guidelines. 715/6693 (10.7%) MINDACT patients had HR+, HER2-, T1abN0 breast cancer and were included in this analysis. All were clinically low-risk, 124/715 (17.3%) were genomic high-risk. For genomic high-risk tumors, 8-year distant metastasis-free survival (DMFS) was 92.9% (95% CI 86.2-96.4%) compared to 95.0% (95% CI 92.8-96.6%) for genomic low-risk tumors. For genomic high-risk tumors treated with or without chemotherapy, 8-year DMFS was 89.2% (95% CI 73.6-95.8%) and 94.1% (95% CI 82.9-98.1%), respectively. For genomic low-risk tumors, the 8-year DMFS and disease-free survival (DFS) were 96.1% (95% CI 93.4-97.6%) and 89.3% (95% CI 85.5-92.2%) when treated with endocrine therapy and 92.9% (95% CI 87.9-95.9%) and 79.4% (95% CI 72.5-84.8%) without. In conclusion, although the number of randomized patients is small, patients with small, genomic high-risk breast cancer did not seem to derive benefit from chemotherapy. Endocrine therapy was associated with improved outcomes even in genomic low-risk breast cancers.

Chemotherapy frequently causes debilitating gastrointestinal symptoms, which are inadequately managed by current treatments. Recent research indicates the gut microbiome plays a role in the pathogenesis of these symptoms. The current study aimed to identify pre-chemotherapy microbiome markers that predict gastrointestinal symptom severity after breast cancer chemotherapy. Fecal samples, blood, and gastrointestinal symptom scores were collected from 59 breast cancer patients before, during, and after chemotherapy. Lower pre-chemotherapy microbiome alpha diversity and abundance of specific microbes (e.g., Faecalibacterium) predicted greater chemotherapy-induced gastrointestinal symptoms. Notably, tumor and diet characteristics were associated with lower pre-chemotherapy alpha diversity. Lower baseline alpha diversity also predicted higher chemotherapy-induced microbiome disruption, which was positively associated with diarrhea symptoms. The results indicate certain cancer patients have lower microbiome diversity before chemotherapy, which is predictive of greater chemotherapy-induced gastrointestinal symptoms and a less resilient microbiome. These patients may be strong candidates for pre-chemotherapy microbiome-directed preventative interventions (e.g., diet change).

Trastuzumab-deruxtecan (T-DXd) has demonstrated intracranial efficacy; however, safety and efficacy data remains limited with stereotactic radiosurgery (SRS). A multi-institutional review was performed with HER2+ or HER2-low metastatic breast cancer treated with T-DXd and SRS for active brain metastases. We identified 215 lesions treated over 48 SRS courses in 34 patients. Median follow up from T-DXd initiation was 13.9 months. The cumulative incidence of symptomatic radiation necrosis at 24 months per lesion was 2.1% and per patient 11%. The 12-month LC was 97%. HER2-low was associated with worse distant intracranial control (DIC) (adjusted HR 2.5, 95% CI 1.1-5.6, p = 0.03) and worse systemic progression free survival (PFS) (HR 4.1, 95% CI 1.6-10.7, p = 0.004). Concurrent SRS and T-DXd has excellent local control, without an increased risk of radiation necrosis. HER2-low disease is associated with worse systemic PFS and DIC with T-DXd compared to HER2+.

With the incorporation of cyclin-dependent kinase inhibitors in early breast cancer (BC), a better identification of biomarkers is needed. The PROMETEO II trial aimed to evaluate the antitumor activity of palbociclib plus letrozole and to identify response biomarkers in patients with operable HR+/HER2- BC and residual disease after neoadjuvant chemotherapy (NAC). The primary endpoint was the rate of complete cell cycle arrest (CCCA), centrally determined by Ki67 ≤ 2.7% at surgery. A comprehensive translational analysis was conducted. At surgery, the CCCA rate was 59.1%, with a 44.2% decrease in Ki67 from the end of NAC. Changes in intrinsic subtypes occurred in 48% of patients, with proliferation genes suppressed, and immune genes more upregulated in tumors with CCCA. Overall, 14% of tumors were classified as PD-L1 after palbociclib. Nine patients experienced grade 3 adverse events (AEs). Palbociclib showed an anti-proliferative effect, with increased immune infiltration in residual tumors with CCCA.Trial registration: Palbociclib Plus Letrozole in Hormone Receptor Positive Residual Disease After Neoadjuvant Chemotherapy (PROMETEO II) ClinicalTrial.gov number NCT04130152. Study registration; October 17, 2019.

This study explores differences in immune cell (IC) composition and spatial distribution between triple-negative breast cancer (TNBC) and hormone receptor-positive, HER2-negative breast cancer (HR + HER2-BC) in high-TIL (≥60%) cases, focusing on PD-L1 status. Using multiplex immunofluorescence on resected tumor tissues from 18 TNBC and 14 HR + HER2-BC cases, we analyzed IC types (CD20, CD8, CD4, FOXP3) and their spatial interactions. TNBC showed a unique IC composition characterized by a higher proportion of CD8 + IC (stroma: 27% vs 17%, p < 0.001; tumor: 54% vs 31%, p < 0.001) and CD4 + FOXP3 + IC (stroma: 3.9% vs 3.0%, p = 0.036), compared to HR + HER2-BC. Notably, PD-L1 positive TNBC cases demonstrated denser infiltration CD4 + FOXP3 + IC in the stromal region compared to HR + HER2-BC (146.4 ± 67.1/mm vs 114.3 ± 146.9/mm, p = 0.036), along with pronounced IC clustering near TC. Both tumor subtypes displayed varied IC compositions based on PD-L1 status. In conclusion, IC composition and spatial distribution in high-TIL TNBC and HR + HER2-BC significantly differ, influenced by PD-L1 status.

Circulating tumor cells (CTCs) have been extensively studied in breast cancer (BC), with large studies establishing CTCs as a robust prognostic biomarker in early and metastatic breast cancer (MBC). Several phase II and phase III trials have investigated the clinical utility of CTCs in BC. Here, we outline the current landscape for the use of CTCs in the clinic at different stages of BC, focusing first on early BC, then on MBC, with a particular focus on interventional clinical trials based on CTCs.

Patients with HER2(+) early breast cancer (EBC) receiving neoadjuvant systemic therapy (NAST) have poorer outcomes if they have residual disease (RD). We analyzed IDFS and brain metastasis (BM) rates in patients with HER2(+) EBC treated with NAST and report the outcomes of patients with HER2(-) RD. Patients with HER2(+) EBC who received NAST between 1 Jan 2019 and 31 Jan 2022 were reviewed. IDFS was defined as the time from surgery until first occurrence of invasive breast cancer recurrence, distant recurrence, or death from any cause. The total cohort was 594 patients. pCR (ypT0/isN0) was achieved in 325(55%) and RD was seen in 269(45%) patients. In 269 patients with RD, 45(17%) did not have HER2 retesting and were excluded. In the remaining 224 patients, 143(64%) were HER2(+) and 81(36%) were HER2(-). With a median follow up of 24 months, 8 patients developed BM at initial recurrence, 4/325(1.2%) with pCR and 4/143(2.8%) with HER2(+) RD. IDFS events occurred in 22/594(3%) patients; 14/269(5%) in RD and 8/325(2%) in pCR (p = 0.04). There was no difference in IDFS between 9/143(6%) patients with HER2(+) RD or 5/81(6%) with HER2(-) RD (p = 0.10). Patients with RD had higher IDFS events than those with pCR. In those with RD, 36% lost HER2(+) status; IDFS events appeared similar in those with HER2(+) RD versus those with HER2(-) RD. The BM events seen in those with RD and pCR highlights the need for more effective therapy in NAST and adjuvant setting to minimize BM risk.