Exploring the Clinical Impact of RANK Pathway Inhibition in Advanced Breast Cancer: Insights From a Retrospective Study on CDK4/6 Inhibitors and Antiresorptive Therapy.

BACKGROUND AND OBJECTIVE: Breast cancer (BC) remains a significant health concern, particularly in advanced stages where the prognosis is poor. The combination of endocrine therapy (ET) with cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) has improved outcomes for advanced BC (aBC) patients. However, resistance to CDK4/6i remains a challenge, with no validated biomarkers to predict response. The receptor activator of the nuclear factor-kB (RANK) pathway has emerged as a key player in aBC, particularly in luminal BC. RANK overexpression has been associated with aggressive phenotypes and resistance to therapy. In view of these findings, we proceeded to investigate the potential involvement of the RANK pathway in luminal BC resistance to CDK4/6i. The objective was to evaluate the effectiveness of denosumab in increasing overall survival (OS) and progression-free survival (PFS). METHODS: In this retrospective analysis, 158 BC patients with bone metastases were included. Patients with human epidermal growth factor receptor-2 (HER2)-negative and hormone receptor-positive BC who received palbociclib or ribociclib in addition to antiresorptive medication were included. Patients received either denosumab or zoledronic acid (ZA) therapy. The primary endpoint was OS, with PFS as a secondary endpoint. RESULTS: Although the PFS and OS of denosumab were better than ZA in this study, it did not show a significant difference between the two drugs. Meanwhile, mOS was not achievable in patients in the denosumab group, while it was 34.1 months in patients in the ZA group. The hazard ratio (HR) showed a significant improvement for the denosumab group in patients under 60 of age (HR: 0.33, p<0.01), patients with a score of 1 HER2 overexpression (HR: 0.09, p=0.01), and patients with resistant endocrine (HR: 0.42, p=0.02) compared to ZA. CONCLUSION: This study highlights the potential clinical relevance of the RANK pathway in BC treatment, and our findings suggest that denosumab may offer significant benefits in terms of PFS and OS for certain subgroups, particularly those with HER2 scores of 1, patients under 60, and those with endocrine-resistant BC. In conclusion, considering that RANK pathway status may be a predictive biomarker for CDK4/6i treatment and may cause treatment resistance, our results demonstrate the clinical relevance of the combination of CDK4/6i + ET with RANKL inhibition.

The Effect of C-Reactive Protein/Lymphocyte Ratio (CLR) on PFS in Metastatic Breast Cancer Patients Treated with CDK4/6 Inhibitors: A Novel Biomarker.

Objective: Hormone positive breast cancer is a tumor with high mortality. Combining antihormonal therapy with cyclin dependent kinase 4/6 inhibitors (CDK4/6i) has resulted in longer survival. The effect of inflammatory parameters such as c-reactive protein and c-reactive protein/lymphocyte ratio (CLR) on efficacy and survival in CDK4/6i treatment is unknown. In our study, we aimed to investigate the role of CLR and some parameters in predicting progression-free survival (PFS) with CDK4/6i. Methods: This retrospective cohort study included 78 patients with denovo and recurrent metastatic breast cancer treated with CDK4/6i. Cut off values for the prediction of mortality by various numerical parameter scores were performed by ROC Curve analysis. The effect of clinical variables, inflammatory and histopathological parameters on survival was analyzed by Kaplan-Meier method. Results: Neutrophil/lymphocyte ratio (NLR) and CLR were statistically significant in predicting mortality (p < 0.05). Ki67 and CLR were correlated with PFS. Age and CLR were correlated with OS (p < 0.05). CLR was statistically significant for both PFS (p = 0.022) and OS (p = 0.006). Conclusion: In patients with metastatic hormone-positive breast cancer using CDK4/6i, low CLR and low Ki67 were correlated with longer PFS duration.

Role of Cyclin-Dependent Kinase 4/6 in Metastatic Breast Cancer: Real-World Data From a Tertiary Care Institute in Eastern India.

Introduction CDK4/6 inhibitors currently approved for patients with hormone-receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) metastatic breast cancer include palbociclib, ribociclib, and abemaciclib. This study aims to report on the treatment outcomes and real-world data regarding the use of CDK4/6 inhibitors in the treatment of ER+/HER2- metastatic breast cancer at a tertiary care institute in Eastern India. Materials and methods The present study is a retrospective analysis of data from patients with metastatic HR+/HER2- breast cancer who were treated with CDK4/6 inhibitors at a tertiary care institute in Eastern India between 2015 and 2022. The data were collected from online records in the departmental files and analyzed for the primary baseline characteristics of the patients, tumors, and response rates, including partial response (PR), complete response (CR), progressive disease (PD), and stable disease (SD), as defined by the Response Evaluation Criteria in Solid Tumors (RECIST) criteria version 1.1. The treatment administered, progression-free survival (PFS), and toxicity were also evaluated. Results From 2015 to 2022, 24 eligible patients were treated with CDK4/6 inhibitors for metastatic HR+/HER2- breast cancer. The average duration of follow-up was 25 months. Out of the 24 patients, 15 (62.5%) were taking Tab. ribociclib, six (25%) were taking Tab. palbociclib, and three (12.5%) were taking Tab. abemaciclib. CDK4/6 was used as a first-line therapy for 16 patients, while eight patients received it as a second-line treatment. Out of the total number of patients, six (25%) had stable disease, 13 (54.2%) had a partial response, and four (16.7%) had progressive disease. In total, of the eligible patients, five (20.8%) had grade I neutropenia, seven (29.2%) had grade II neutropenia, and four (16.7%) had grade III neutropenia. At five years, the PFS rate estimated by the Kaplan-Meier method was 50% (95% CI: 47.89-69.31). Conclusion Ribociclib and palbociclib have improved PFS in patients with metastatic HR+/HER2- breast cancer. Both drugs have well-tolerated toxicity, allowing patients to continue taking them for an extended period of time. CDK4/6 inhibitors have a higher response rate than the other agents.

Beliefs about medicines' association with endocrine therapy adherence in early breast cancer survivors in Croatia.

This observational, cross-sectional study conducted at the University Hospital Centre Zagreb (UHC Zagreb) aimed to explore patients' beliefs about adjuvant endocrine therapy (AET) as well as their association with non-adherence and sociodemographic and clinical factors. Out of 420 early breast cancer (BC) patients included in the study, 79.5 % perceived AET necessary and important for their health, as measured by the Belief About Medicines Questionnaire (BMQ), with the mean necessity score (20.4 ± 3.68) significantly higher than the mean concerns score (13 ± 4.81) (p < 0.001). Based on the Medication Adherence Report Scale (MARS-5), 44.4 % (n = 182) of the participants were non-adherers, out of which 63.2 % (n = 115) were unintentional and 36.8 % (n = 67) intentional non-adherers. Significantly higher concern beliefs were found among patients that were younger (p < 0.001), employed (p < 0.001), intentionally non-adherent to AET (p = 0.006), had a lower body-mass index (p = 0.005) and a higher level of education (p < 0.001), were premenopausal at the time of diagnosis (p < 0.001), taking tamoxifen treatment (p = 0.05) and receiving ovarian suppression (p < 0.001). Younger patients should be recognized as being at risk of non-adherence as they hold greater concern beliefs about medicines.

Oncotype Dx Score, HER2 Low Expression, and Clinical Outcomes in Early-Stage Breast Cancer: A National Cancer Database Analysis.

BACKGROUND: The interaction between HER2-low expression, oncotype recurrence score (RS), and their influence on the prognosis of HR+/HER2- breast cancer (BC) is not very well studied. METHODS: We conducted a retrospective cohort study of patients diagnosed with resectable HER2-low and HER2-zero BC from the National Cancer Database. The primary outcome was overall survival (OS), and the association of RS with the clinical outcomes in HR+/HER2- BC was analyzed as an exploratory endpoint. RESULTS: The distribution of RS was comparable between HER2-low and HER2-zero groups; however, the RSs of HER2-low tumors were more likely to be 16-25. Women with HER2-low tumors had longer 5-year OS than women with HER2-zero tumors in the HR-negative (84.3% vs. 83.9%; p < 0.001, HR: 0.87 (0.84-0.90), p < 0.001) but not in the HR-positive group (94.0% vs. 94.0%; p = 0.38, HR: 0.97 (0.95-0.99), p = 0.01). The survival advantage was observed in patients who received adjuvant/neoadjuvant chemotherapy (p-interaction (chemo vs. no chemo) < 0.001). Among those who received adjuvant chemotherapy in the group with higher RSs (26-100), those with HER2-low BC had higher 5-year OS than HER2-zero BC. CONCLUSIONS: Resectable HER2-low BC had a better prognosis than HER2-zero BC. Among those who received adjuvant chemotherapy in the higher oncotype RS group, those with HER2-low tumors had better survival.

Evaluating everolimus for the treatment of breast cancer.

INTRODUCTION: Everolimus is an oral drug that inhibits mTOR with immunosuppressive and antiproliferative characteristics. It is commonly used in association with exemestane in hormone receptor (HR)-positive advanced breast cancer (ABC). AREAS COVERED: The current review summarizes the publications relating to everolimus from clinical research in breast cancer. Everolimus showed treatment efficacy and an acceptable safety tolerance with the prevention of side effects in Phase II/III studies. BOLERO-2 study showed a progression-free survival improvement in patients with HR - positive ABC previously treated with aromatase inhibitors (AI) and leading to its acceptance in this indication. The absence of a post-CDK4/6 inhibitor (CDK4/6i.) study and the arrival of new drugs may raise questions about its current place in the therapeutic strategy. EXPERT OPINION: Everolimus is relevant in the management of HR - positive ABC. Because of its efficacy, acceptable tolerability and the absence of drugs that have shown a greater benefit, it remains a second-line treatment option in HR-positive, HER2 negative (score 0) patients without BRCA mutation or visceral crisis and can be discussed with fulvestrant in second line after CDK4-6i. It is likely that within 5 years this treatment will be replaced in second-line HR-positive breast cancer by new emerging treatments: drug-conjugated antibodies, tyrosine kinase inhibitors or immunotherapy in combination with chemotherapy.

Androgen Receptor in Hormone Receptor-Positive Breast Cancer.

Breast cancer subtypes expressing hormone receptors (HR+ BCa) have a good prognosis and respond to first-line endocrine therapy (ET). However, the majority of HR+ BCa patients exhibit intrinsic or acquired ET resistance (ET-R) and rapid onset of incurable metastatic BCa. With the failure of conventional ET, limited targeted therapy exists for ET-R HR+ BCa patients. The androgen receptor (AR) in HR-negative BCa subtypes is emerging as an attractive alternative target for therapy. The AR drives Luminal AR (LAR) triple-negative breast cancer progression, and LAR patients consistently exhibit positive clinical benefits with AR antagonists in clinical trials. In contrast, the function of the AR in HR+ BCa is more conflicting. AR in HR+ BCa correlates with a favorable prognosis, and yet, the AR supports the development of ET-R BCa. While AR antagonists were ineffective, ongoing clinical trials with a selective AR modulator have shown promise for HR+ BCa patients. To understand the incongruent actions of ARs in HR+ BCa, the current review discusses how the structure and post-translational modification impact AR function. Additionally, completed and ongoing clinical trials with FDA-approved AR-targeting agents for BCa are presented. Finally, we identify promising investigational small molecules and chimera drugs for future HR+ BCa therapy.

Impact of body mass index on the efficacy of aromatase inhibitors in patients with metastatic breast cancer.

PURPOSE: Higher levels of estrogen in obese patients may lead to incomplete inhibition by aromatase inhibitors (AIs). The aim of this study was to determine the impact of body mass index (BMI) on efficacy of AIs in patients with metastatic hormone receptor (HR)-positive breast cancer (BC). METHODS: We performed a retrospective chart review of all female patients with metastatic HR-positive BC on an AI in first- or second-line settings and seen at our academic institution between 2001 and 2020. The primary endpoint was progression-free survival (PFS), defined as the time from start of AI to disease progression or death from any cause. RESULTS: We identified 219 patients who had received an AI in the first- or second-line settings for metastatic HR-positive BC and with documented information on BMI. Of the 219 patients, 56% (123) had a low BMI (defined as < 27 kg/m2) and 44% (96) had a high BMI (≥ 27 kg/m2). The median PFS was 21.9 months (95% CI 14.5 to 28.4) in the low BMI group versus 20.2 months (95% CI 14.3 to 27.5) in the high BMI group (p = 0.73). CONCLUSION: While BMI influences efficacy of AIs in the adjuvant setting, our results suggest that in the metastatic setting, BMI may not impact the efficacy of AIs. This discrepancy could be due to other differences in disease characteristics that make complete aromatase inhibition more important in the adjuvant setting when disease burden is the lowest.

The Role of Ki67 in Evaluating Neoadjuvant Endocrine Therapy of Hormone Receptor-Positive Breast Cancer.

Ki67 is a proliferation marker. It has been proposed as a useful clinical marker for breast cancer subtype classification, prognosis, and prediction of therapeutic response. But the questionable analytical validity of Ki67 prevents its widespread adoption of these measures for treatment decisions in breast cancer. Currently, Ki67 has been tested as a predictive marker for chemotherapy using clinical and pathological response as endpoints in neoadjuvant endocrine therapy. Ki67 can be used as a predictor to evaluate the recurrence-free survival rate of patients, or its change can be used to predict the preoperative "window of opportunity" in neoadjuvant endocrine therapy. In this review, we will elaborate on the role of Ki67 in neoadjuvant endocrine therapy in breast cancer.

Population-based analysis of non-operative management and treatment patterns in older women with estrogen receptor-positive breast cancer.

PURPOSE: To examine the proportion of older women with ER + HER2- breast cancer receiving non-operative management versus surgery, and to evaluate the use of axillary staging and adjuvant radiation in this population. METHODS: We queried the SEER database to identify all women aged 70 years or older with stage I-III ER + HER2- invasive breast cancer diagnosed between 2010 and 2016. We evaluated trends in non-operative management, breast surgery, axillary staging, and adjuvant radiation according to age at diagnosis. RESULTS: We identified 57,351 older women with ER + HER2- disease. Overall, 3538 (6.2%) of the cohort underwent non-operative management, 38,452 (67.0%) underwent breast-conserving surgery (BCS), and 15,361 (26.8%) underwent mastectomy. The proportion of patients undergoing non-operative management increased from 2.8% among 70-74-year-old women to 30.1% in those ≥ 90 years old (p < 0.001). In 53,813 women who underwent surgery, 36,850 (68.5%) underwent sentinel lymph node biopsy, while 10,861 (20.2%) underwent axillary lymph node dissection. Subgroup analysis of 29,032 older women undergoing BCS for stage I ER + HER2- breast cancer revealed a 14.2% rate of omission of axillary staging, increasing from 5.3% in those 70-74 years to 67.6% in those ≥ 90 years old (p < 0.001). Receipt of adjuvant radiation occurred in 63.3% of older women following BCS and 18% post-mastectomy, with similar trends towards omission in older age groups. CONCLUSION: Primary breast surgery remains the dominant management strategy for the majority of older women with ER + HER2- breast cancer. Omission of axillary staging and adjuvant radiation are used in a minority of eligible women undergoing breast conservation for early-stage disease.

A comparative study between Ki67 positive versus Ki67 negative females with breast cancer: Cross sectional study.

INTRODUCTION: The prognosis of breast cancer depends on several clinical and pathological parameters most importantly the clinical stage, other factors predicting the outcome are hormone receptors like estrogen and progesterone receptors. Expression of Ki67 also have been shown to affect the outcome. PATIENTS AND METHODS: This retrospective study included 278 female patients diagnosed and operated for breast cancer. Patients were grouped into 2 groups according to the expression of Ki67 to those with positive and those with negative expression. Both groups were compared for differences. RESULTS: The mean age was 48.61 years and the right breast was the commonest affected side, the mean tumor size was 34 mm, 70% had axillary LN involvement, 50% had intermediate tumor grade, and 85.6% had no recurrence. Most patients had stage IIA, IIB, and IIIA, 67.6% had positive expression of Ki67 and had a significant correlation with the tumor grade, tumor necrosis, and ER expression (P values 0.001, 0.047, and 0.002) respectively, while the correlation was negative with recurrence, axillary LN involvement, TNM stage, site of the tumor, age, tumor size, PR and HER-2 receptor (P values 0.476, 0.971, 0.509, 0.405, 0.122, 0.994, 0.892, and 0.418) respectively. CONCLUSION: Most patients with breast cancer have positive expression of Ki67 which has a positive correlation with tumor grade, the presense of necrosis inside the tumor and estrogene receptor status. This marker is directly related with higher degrees of tumor agressiveness and may be useful in modulating different treatment modalities.

Rationally Designed Ruthenium Complexes for Breast Cancer Therapy.

Since the discovery of the anticancer potential of ruthenium-based complexes, several species were reported as promising candidates for the treatment of breast cancer, which accounts for the greatest number of new cases in women every year worldwide. Among these ruthenium complexes, species containing bioactive ligand(s) have attracted increasing attention due to their potential multitargeting properties, leading to anticancer drug candidates with a broader range of cellular targets/modes of action. This review of the literature aims at providing an overview of the rationally designed ruthenium-based complexes that have been reported to date for which ligands were carefully selected for the treatment of hormone receptor positive breast cancers (estrogen receptor (ER+) or progesterone receptor (PR+)). In addition, this brief survey highlights some of the most successful examples of ruthenium complexes reported for the treatment of triple negative breast cancer (TNBC), a highly aggressive type of cancer, regardless of if their ligands are known to have the ability to achieve a specific biological function.

Utilization, duration, and outcomes of neoadjuvant endocrine therapy in the United States.

PURPOSE: To evaluate if real-world utilization of neoadjuvant endocrine therapy (NET) is associated with similar rates of response and breast conservation surgery (BCS) compared to neoadjuvant chemotherapy (NAC). METHODS: Our population-based assessment used the National Cancer Data Base to identify women diagnosed with stage II-III, hormone receptor (HR)-positive BC who underwent surgery and received endocrine therapy from 2004 to 2014. Women were categorized by receipt of NET, NAC or no neoadjuvant therapy. We used logistic regression to assess differences in outcomes between therapies using inverse propensity score weighting to adjust for potential selection bias. RESULTS: In our sample of 211,986 women, 6584 received NET, 52,310 received NAC, and 153,092 did not receive any neoadjuvant therapy. After adjusting for multiple relevant covariates and cofounders, there was no significant difference between NET and NAC with regard to BCS [odds ratio (OR) 0.91; 95% confidence interval (CI) (0.82-1.01)]; however, women who received NET were significantly less likely to achieve pCR [OR 0.34; 95% CI (0.23-0.51)] or a decrease in T stage [OR 0.39; CI (0.34-0.44)] compared to women treated with NAC. Patients who received NET for ≥ 3 months had higher odds of BCS (OR 1.59; 95% CI 1.46-1.73) and downstaging (OR 1.79; 95% CI 1.63-1.97) compared to patients who did not receive neoadjuvant therapy. CONCLUSIONS: Women who received NET had similar rates of BCS compared to women who received NAC. Those who received NET for longer treatment durations had increased odds of BCS and downstaging compared to women who did not receive neoadjuvant therapy.

Relationship of pathological features and a 21 gene expression assay in younger versus older women with node-negative endocrine receptor-positive breast cancer.

PURPOSE: Determining the need for adjuvant chemotherapy in estrogen receptor (ER)+ disease can be influenced by pathological characteristics and gene expression assays [i.e., Oncotype Dx recurrence scores (RSs)]. The primary objective of this study is to investigate the relationship between the RSs and pathological markers in younger (< 50) versus older (≥ 50) women with early-stage node-negative ER+ breast cancer. METHODS: This was a single academic-center retrospective cohort study. Subjects who underwent Oncotype gene expression testing were retrospectively and sequentially identified. 436 Subjects were identified of which 344 were eligible for analysis (133 younger subjects < 50 years of age, and 211 older subjects ≥ 50 years). Pathological data assessed included the progesterone receptor (PR), histological grade (grade), Ki-67, and P53. A multivariable regression analysis was performed using age, PR, and grade as predictor variables for RS. Adjusted R2 was determined. To investigate the primary objective, subjects were stratified based on age, PR, and grade status in that sequence. Within each tumor subtype as determined by PR and grade statuses, the RSs in the younger versus older age group were compared using Student's t-test and the differences in the 95% confidence interval limits in RS means calculated. Age influence on adjuvant chemotherapy recommendation was also assessed by stratifying subjects based on age (< 50 vs. ≥ 50) and then by RS risk group (≤ 10, 11-25, ≥ 26). Subsequently, the proportions of younger versus older subjects within identical RS risk groups who were explicitly advised by their oncologist to proceed with chemotherapy as documented in their electronic health records were compared using χ2 test. RESULTS: Based on the multivariable regression analysis, the adjusted R2 was 0.229232 and RS was found to be independent of age (p = 0.7169). Between younger and older subjects with tumors with similar PR and grade pathological features, the differences in the RS were insignificant (p > 0.05). Chemotherapy was recommended in younger versus older women, in 0% when the RS was ≤ 10, 39% and 40% when the RS was 11-25 (p = 0.82), and 100% and 98% when the RS was ≥ 26 (p = 0.51), respectively. CONCLUSIONS: The relationship between pathological features and RS is consistent irrespective of age; therefore, models predicting RS may be applicable irrespective of age.

Impact of CALGB 9343 Trial and Sociodemographic Variation on Patterns of Adjuvant Radiation Therapy Practice for Elderly Women (≥70 Years) with Stage I, Estrogen Receptor-positive Breast Cancer: Analysis of the National Cancer Data Base.

BACKGROUND: The Cancer and Leukemia Group B (CALGB) 9343 trial demonstrated that adjuvant radiation therapy (RT) can be omitted in women 70 years or older, with small (≤2 cm), negative lymph nodes, estrogen receptor (ER)-positive breast cancer. We examined whether RT usage following the CALGB publication had decreased over time and evaluated sociodemographic and clinical factors associated with RT omission. MATERIALS AND METHODS: From the National Cancer Data Base, we analyzed a cohort of 120,308 women aged 70 years or older with stage I, ER-positive breast cancer who underwent lumpectomy. Patients were classified into two groups based on the time of CALGB 9343 publication: (i) pre-CALGB (up to 2004), and (ii) post-CALGB (2005-2012). Clinicopathological and sociodemographic variables were compared between pre- and post-CALGB groups. Chi-square and multivariable logistic regression were employed, with the omission of adjuvant RT as the primary outcome in the regression analysis. RESULTS: Radiation therapy usage decreased by 4.1% after CALGB publication (on average 71.6% pre-CALGB vs. 67.5% post-CALGB; p<0.0001). Almost one-third of women aged ≥85 years received RT in the post-CALGB group. In a multivariable model, the variables significantly associated with increased odds for omission of RT in the post-CALGB group were: advanced age, African-American, increased great circle distance, therapy under academic research program, residents of East South-Central region, living in a rural population <2,500 not adjacent to a metropolitan area, low income level, Medicaid recipients, high comorbidity index, small tumor, well-differentiated histology, residual tumor, and lack of receipt of chemotherapy and anti-hormonal therapy. CONCLUSION: During the study period, the CALGB trial publication had a minimal impact on the rate of adjuvant RT use among elderly women with small, ER-positive breast cancers. Significant variation in RT usage existed across sociodemographic strata.

Endocrine therapy adherence: a cross-sectional study of factors affecting adherence and discontinuation of therapy.

BACKGROUND/AIMS: Adjuvant endocrine therapy for at least 5 years improves oncological outcomes in oestrogen receptor-positive breast cancer. Adherence rates to prescribed endocrine therapy are low and the search for modifiable causes of this continues. The aim of this study was to assess adherence rates in an Irish cohort of breast cancer patients prescribed adjuvant endocrine therapy and to assess modifiable factors associated with suboptimal adherence. METHODS: A cross-sectional anonymous survey was performed on 261 patients currently prescribed endocrine therapy. Data were collected regarding demographics, treatment, social and emotional factors and medication side effects. Each patient completed a medication adherence score and provided information about discontinuation of therapy and reasons for same. RESULTS: Only 67.8 % of patients assessed demonstrated complete medication adherence on the medication adherence scale. Twenty-nine patients (10.9 %) permanently stopped taking their prescribed endocrine therapy. Suboptimal adherence was more likely in younger patients (p < 0.001), those in employment (p = 0.005), those who experienced side effects (p = 0.006), those who perceived themselves to have low levels of emotional support (p < 0.001) and those who use the internet to read about their illness (p = 0.003). CONCLUSIONS: Endocrine therapy adherence is suboptimal in almost one-third of patients in our cohort. Appropriate assessment and management of side effects and negative emotions, combined with direction of patients to accurate internet sources of information, could help improve endocrine therapy adherence in women with early-stage breast cancer.