SNP of Aromatase Predict Long-term Survival and Aromatase Inhibitor Toxicity in Patients with Early Breast Cancer: A Biomarker Analysis of the GIM4 and GIM5 Trials.

PURPOSE: In estrogen receptor-positive (ER+) breast cancer, single-nucleotide polymorphisms (SNP) in the aromatase gene might affect aromatase inhibitors (AI) metabolism and efficacy. Here, we assessed the impact of SNP on prognosis and toxicity of patients receiving adjuvant letrozole. EXPERIMENTAL DESIGN: We enrolled 886 postmenopausal patients in the study. They were treated with letrozole for 2 to 5 years after taking tamoxifen for 2 to 6 years, continuing until they completed 5 to 10 years of therapy. Germline DNA was genotyped for SNP rs4646, rs10046, rs749292, and rs727479. Log-rank test and Cox model were used for disease-free survival (DFS) and overall survival (OS). Cumulative incidence (CI) of breast cancer metastasis was assessed through competing risk analysis, with contralateral breast cancer, second malignancies and non-breast cancer death as competing events. CI of skeletal and cardiovascular events were assessed using DFS events as competing events. Subdistribution HR (sHR) with 95% confidence intervals were calculated through Fine-Gray method. RESULTS: No SNP was associated with DFS. Variants rs10046 [sHR 2.03, (1.04-2.94)], rs749292 [sHR 2.11, (1.12-3.94)], and rs727479 [sHR 2.62, (1.17-5.83)] were associated with breast cancer metastasis. Three groups were identified on the basis of the number of these variants (0, 1, >1). Variant-based groups were associated with breast cancer metastasis (10-year CI 2.5%, 7.6%, 10.7%, P = 0.035) and OS (10-year estimates 96.5%, 93.0%, 89.6%, P = 0.030). Co-occurrence of rs10046 and rs749292 was negatively associated with 10-year CI of skeletal events (3.2% vs. 10%, P = 0.033). A similar association emerged between rs727479 and cardiovascular events (0.3% vs. 2.1%, P = 0.026). CONCLUSIONS: SNP of aromatase gene predict risk of metastasis and AI-related toxicity in ER+ early breast cancer, opening an opportunity for better treatment individualization.

Naringenin improves ovarian health by reducing the serum androgen and eliminating follicular cysts in letrozole-induced polycystic ovary syndrome in the Sprague Dawley rats.

Polycystic ovary syndrome (PCOS) is most common in women of reproductive age, giving rise to androgen excess and anovulation, leading to infertility and non-reproductive complications. We explored the ameliorating effect of naringenin in PCOS using the Sprague Dawley (SD) rat model and human granulosa cells. Letrozole-induced PCOS rats were given either naringenin (50 mg/kg/day) alone or in combination with metformin (300 mg/kg/day), followed by the estrous cycle, hormonal analysis, and glucose sensitivity test. To evaluate the effect of naringenin on granulosa cell (hGC) steroidogenesis, we treated cells with naringenin (2.5 µM) alone or in combination with metformin (1 mM) in the presence of forskolin (10 µM). To determine the steroidogenesis of CYP-17A1, -19A1, and 3ßHSD2, the protein expression levels were examined. Treatment with naringenin in the PCOS animal groups increased ovulation potential and decreased cystic follicles and levels of androgens. The expression levels of CYP-17A1, -19A1, and 3ßHSD2, were seen restored in the ovary of PCOS SD rats' model and in the human ovarian cells in response to the naringenin. We found an increased expression level of phosphorylated-AKT in the ovary and hGCs by naringenin. Naringenin improves ovulation and suppress androgens and cystic follicles, involving AKT activation.

Ameliorative effect of sesame oil on experimentally induced polycystic ovary syndrome: A cross-link between XBP-1/PPAR-1, regulatory proteins for lipogenesis/steroids.

Polycystic ovary syndrome (PCOS) is a mixed endocrine/metabolic/reproductive disorder in women of reproductive age. Sesame oil (SO) contains sesame lignans & vitamin E with broad-spectrum antioxidant and anti-inflammatory effects. This study investigates the ameliorative effect of SO on experimentally induced PCOS and elucidates the possible molecular mechanisms with a deeper focus on the different signaling pathways involved. The study was carried out on 28 nonpregnant female Wister albino rats that were divided into four equal groups; Group I (control group) received oral 0.5% wt/vol carboxymethyl cellulose daily. Group II (SO group): orally administered SO (2 mL/kg body wt./day) for 21 days. Group III (PCOS group) received letrozole daily, 1 mg/kg, for 21 days. Group IV (PCOS + SO group): concomitantly administered letrozole and SO for 21 days. The serum hormonal and metabolic panel and the homogenate ATF-1, StAR, MAPK, PKA, and PI3K levels of the ovarian tissue were calorimetrically evaluated. However, endoplasmic reticulum (ER) stress was evaluated by ovarian XBP1 and PPAR-γ messenger RNA expression level using the qRT-PCR technique. Ovarian COX-2 was detected immunohistochemically. The results suggest that SO-treated PCOS rats showed a significantly improved hormonal, metabolic panel, inflammatory, and ER stress status with concomitant decreases in ATF-1, StAR, MAPK, PKA, and PI3K in ovarian rats compared to the correspondent values in PCOS without treatment. CONCLUSIONS: The protective effects of SO against PCOS are triggered by ameliorating regulatory proteins of ER stress, lipogenesis, and steroidogenesis through the PI3K/PKA and MAPK/ERK2 signaling cascades. SIGNIFICANCE STATEMENT: Polycystic ovary syndrome (PCOS) is the most common mixed endocrine-metabolic dysfunction among women within the reproductive period, with an estimated prevalence of 5%-26% worldwide. Doctors traditionally recommend metformin for PCOS patients. However, metformin is known to be associated with significant adverse effects and contraindications. This work aimed at shedding light on the ameliorative effect of sesame oil (SO), natural polyunsaturated fatty acids-rich oil, on the induced PCOS model. SO proved to have a marvelous effect on the metabolic and endocrine derangements in the PCOS rat model. We hoped to provide a valuable alternative treatment for PCOS patients to avoid the side effects of metformin and to help PCOS patients for whom metformin is contraindicated.

Milicia excelsa (Moraceae) reversed the effects of letrozole-induced aromatase inhibition in rats with polycystic ovary syndrome.

OBJECTIVES: Polycystic ovary syndrome (PCOS) is an endocrinopathy affecting 5-20% of women of childbearing age. There is no single drug for the treatment of PCOS and current therapies have significant side effects. This study evaluated the ability of Milica excelsa to improve PCOS symptoms in rats. METHODS: Induction of PCOS was achieved using letrozole (a reversible aromatase inhibitor; 1 mg/kg; given orally for 21 days). From day 22, PCOS rats received the aqueous extract of M. excelsa roots (14 and 140 mg/kg). Clomiphene citrate (1 mg/kg) was administered to the positive control. The negative and the normal controls received the vehicle (5% DMSO). Treatments were given orally for 7 or 14 days. Vaginal smears were scrutinized daily during the experiment. Body weight was measured hebdomadal. Animals were sacrificed after the two treatment periods for biochemical and histological analyses. RESULTS: Aromatase inhibition caused hyperandrogenism (p<0.001), overweight (p<0.001) and fat accumulation (p<0.001). It also blocked the estrous cycle at the diestrus phase and altered ovarian dynamics as evidenced by the accumulation of cystic (p<0.001) and atretic (p<0.001) follicles. In contrast, M. excelsa induced weight loss (p<0.001), reduction in fat weight (p<0001), and lower serum androgen and LH levels (p<0.001). It also restored the estrous cycle and improved ovarian dynamics by increasing the amount of Graafian follicles (p<0.001) and corpora lutea (p<0.001), and decreasing that of cystic and atretic follicles (p<0.001). CONCLUSIONS: Milica excelsa corrected hyperandrogenism and overweight in PCOS animals, and reduced cyst formation and follicle atresia in their ovaries.

Therapeutic Potential of Selenium Nanoparticles on Letrozole-Induced Polycystic Ovarian Syndrome in Female Wistar Rats.

Polycystic ovarian syndrome (PCOS) is considered the most frequent gynecological endocrine disorder that causes anovulatory infertility. The current study aimed to investigate the potential significance of selenium nanoparticles (SeNPs), an IL-1 inhibitor, in the treatment of letrozole-induced PCOS in rats that satisfied the metabolic and endocrine parameters found in PCOS patients. Letrozole (2 ppm, per orally, p.o.) was given orally to female Wistar rats for 21 days to develop PCOS. After PCOS induction, rats were given SeNPs (25 ppm/day, p.o.), SeNPs (50 ppm/day, p.o.), or metformin (2 ppm/day, p.o.) for 14 days. PCOS was associated with an increase in body weight, ovarian weight, ovarian size, and cysts, as well as an increase in blood testosterone, luteinizing hormone (LH), and insulin, glycaemia, and lipid profile levels. The SeNP administration decreased all of these variables. Furthermore, SeNPs significantly reduced letrozole-induced oxidative stress in the ovaries, muscles, and liver by decreasing elevated levels of malondialdehyde and total nitrite while raising suppressed levels of superoxide dismutase and catalase. SeNPs increased the amounts of the protective proteins Kelch-like ECH-associated protein 1 (Keap-1), nuclear factor erythroid 2-related factor 2 (Nrf2), and OH-1. It was depicted from the study that SeNPs reduce the upregulation of inflammatory cytokines that are interleukin 6 (IL-6), tumour necrosis factor α (TNF-α), and the interleukin 1 (IL-1). Our findings show that SeNPs, through their antioxidant and anti-inflammatory characteristics, alleviate letrozole-induced PCOS.

ΔKi67 proliferation index as independent predictive and prognostic factor of outcome in luminal breast cancer: data from neoadjuvant letrozole-based treatment.

A key tool for monitoring breast cancer patients under neoadjuvant treatment is the identification of reliable predictive markers. Ki67 has been identified as a prognostic and predictive marker in ER-positive breast cancer. Ninety ER-positive, HER2 negative locally advanced breast cancer patients received letrozole (2.5 mg daily) and cyclophosphamide (50 mg daily) with/without Sorafenib (400 mg/bid daily) for 6 months before undergoing surgery. Ki67 expression and tumor size measured with caliber were determined at baseline, after 30 days of treatment and at the end of treatment. Patients were assigned to a clinical response category according to Response Evaluation Criteria in Solid Tumors, both at 30 days and before surgery and further classified as high-responder and low-responder according to the median variation of Ki67 values between biopsy and 30 days and between biopsy and surgery time. The predictive role of Ki67 and its changes with regard to clinical response and survival was analyzed. No differences in terms of survival outcomes emerged between the arms of treatment, while we observed a higher percentage of women with progression or stable disease in arm with the combination containing Sorafenib (20.5% vs 7.1%, p = 0.06). Clinical complete responders experienced a greater overall variation in Ki67 when compared with partial responders and patients with progressive/stable disease (66.7% vs 30.7%, p = 0.009). High responders showed a better outcome than low responders in terms of both disease-free survival (p = 0.009) and overall survival (p = 0.002). ΔKi67 score evaluated between basal and residual tumor at definitive surgery showed to be highly predictive of clinical complete response, and a potential parameter to be used for predicting disease-free survival and overall survival in luminal breast cancer treated with neoadjuvant endocrine-based therapy.

Association of CYP19A1 gene variations with adjuvant letrozole-induced adverse events in South Indian postmenopausal breast cancer cohort expressing hormone-receptor positivity.

PURPOSE: Musculoskeletal adverse events (MS-AEs) and vasomotor symptoms (VMSs) are the major side-effects of newer generation non-steroidal aromatase inhibitor (AI), letrozole. Single-nucleotide polymorphisms (SNPs) in CYP19A1 gene coding for the enzyme aromatase are related to AI treatment-associated adverse drug reactions. Therefore, we aimed to determine whether SNPs in the CYP19A1 gene are associated with adjuvant letrozole-induced 'specific' AEs in postmenopausal hormone receptor-positive (HR+) breast cancer patients. METHODS: Genomic DNA was isolated from 198 HR+ breast cancer patients by the phenol-chloroform method, and eleven SNPs in the CYP19A1 gene were genotyped by TaqMan genotyping assays on the qRT-PCR system. Toxicity was assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 3.0, and the data were analyzed using SPSS v19.0 and Haploview v4.2 statistical software. RESULTS: Subjects carrying the genetic variants of CYP19A1 gene SNP rs700519 had significantly higher odds (OR 2.33; 95% CI [1.29-4.20], P = 0.0057) of MS-AEs under dominant statistical effect. The frequency of the two distinct haplotypes that include the variant allele 'T' at rs700519 locus, H5-GCTATCTGGCG (P = 0.042) and H11-GCTATTGCACG (P = 0.013) were significantly higher in patients with musculoskeletal toxicity than in those without MS-AEs and thus predisposing to MS-AEs. Similarly, H6-GCCAGCTGGCG (P = 0.037) haplotype exhibited higher frequencies in patients presented with VMSs. However, no such association was observed between CYP19A1 genotypes and VMSs. CONCLUSIONS: To the best of our knowledge, this is the first study assessing the impact of CYP19A1 genetic variations with adjuvant letrozole treatment-associated AEs in Indian women. Genetic variations in the CYP19A1 gene is associated with letrozole-induced AEs and warrants further investigation in larger cohorts to validate this finding.

Complementary and alternative medicine and musculoskeletal pain in the first year of adjuvant aromatase inhibitor treatment in early breast cancer patients.

BACKGROUND: Patients with breast cancer (BC) show strong interest in complementary and alternative medicine (CAM), particularly for adverse effects of adjuvant endocrine treatment - e.g., with letrozole. Letrozole often induces myalgia/limb pain and arthralgia, with potential noncompliance and treatment termination. This analysis investigated whether CAM before aromatase inhibitor (AI) therapy is associated with pain development and the intensity of AI-induced musculoskeletal syndrome (AIMSS) during the first year of treatment. PATIENTS AND METHODS: The multicenter phase IV PreFace study evaluated letrozole therapy in postmenopausal, hormone receptor-positive patients with early BC. Patients were asked about CAM use before, 6 months after, and 12 months after treatment started. They recorded pain every month for 1 year in a diary including questions about pain and numeric pain rating scales. Data were analyzed for patients who provided pain information for all time points. RESULTS: Of 1396 patients included, 901 (64.5%) had used CAM before AI treatment. Throughout the observation period, patients with CAM before AI treatment had higher pain values, for both myalgia/limb pain and arthralgia, than non-users. Pain increased significantly in both groups over time, with the largest increase during the first 6 months. No significant difference of pain increase was noted regarding CAM use. CONCLUSIONS: CAM use does not prevent or improve the development of AIMSS. Pain intensity was generally greater in the CAM group. Therefore, because of the risk of non-compliance and treatment discontinuation due to the development of higher pain levels, special attention must be paid to patient education and aftercare in these patients.

Use of letrozole after aromatase inhibitor-based therapy in postmenopausal breast cancer (NRG Oncology/NSABP B-42): a randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: The optimal duration of extended therapy with aromatase inhibitors in patients with postmenopausal breast cancer is unknown. In the NSABP B-42 study, we aimed to determine whether extended letrozole treatment improves disease-free survival after 5 years of aromatase inhibitor-based therapy in women with postmenopausal breast cancer. METHODS: This randomised, double-blind, placebo-controlled, phase 3 trial was done in 158 centres in the USA, Canada, and Ireland. Postmenopausal women with stage I-IIIA hormone receptor-positive breast cancer, who were disease-free after about 5 years of treatment with an aromatase inhibitor or tamoxifen followed by an aromatase inhibitor, were randomly assigned (1:1) to receive 5 years of letrozole (2·5 mg orally per day) or placebo. Randomisation was stratified by pathological node status, previous tamoxifen use, and lowest bone mineral density T score in the lumbosacral spine, total hip, or femoral neck. The primary endpoint was disease-free survival, defined as time from randomisation to breast cancer recurrence, second primary malignancy, or death, and was analysed by intention to treat. To adjust for previous interim analyses, the two-sided statistical significance level for disease-free survival was set at 0·0418. This study is registered with ClinicalTrials.gov, number NCT00382070, is active, and is no longer enrolling patients. FINDINGS: Between Sept 28, 2006, and Jan 6, 2010, 3966 patients were randomly assigned to receive letrozole (n=1983) or placebo (n=1983). Follow-up information was available for 3903 patients for the analyses of disease-free survival. Median follow-up was 6·9 years (IQR 6·1-7·5). Letrozole treatment did not significantly improve disease-free survival (339 disease-free survival events were reported in the placebo group and 292 disease-free survival events were reported in the letrozole group; hazard ratio 0·85, 95% CI 0·73-0·999; p=0·048). 7-year disease-free survival estimate was 81·3% (95% CI 79·3-83·1) in the placebo group and 84·7% (82·9-86·4) in the letrozole group. The most common grade 3 adverse events were arthralgia (47 [2%] of 1933 patients in the placebo group vs 50 [3%] of 1941 patients in the letrozole group) and back pain (44 [2%] vs 38 [2%]). The most common grade 4 adverse event in the placebo group was thromboembolic event (eight [<1%]) and the most common grade 4 adverse events in the letrozole group were urinary tract infection, hypokalaemia, and left ventricular systolic dysfunction (four [<1%] each). INTERPRETATION: After 5 years of aromatase inhibitor-based therapy, 5 years of letrozole therapy did not significantly prolong disease-free survival compared with placebo. Careful assessment of potential risks and benefits is required before recommending extended letrozole therapy to patients with early-stage breast cancer. FUNDING: National Cancer Institute, Korea Health Technology R&D Project, Novartis.

A randomized controlled trial comparing lifestyle intervention to letrozole for ovulation in women with polycystic ovary syndrome: a study protocol.

BACKGROUND: Polycystic ovary syndrome (PCOS) affects between 8 and 18% of women and is the leading cause of female anovulatory infertility. Unfortunately, common treatments for women trying to conceive can be ineffective as well as disruptive or harmful to patients' quality of life. Despite evidence that women with PCOS have expressed the need for alternative fertility treatments, lifestyle interventions incorporating a nutritional plan with supplementation, increased physical activity, and techniques for stress management have not been combined as a program and studied in this population. Literature suggests that each of these individual components can positively influence reproductive hormones and metabolic health. METHODS/DESIGN: This is a randomized controlled trial which will include 240 women diagnosed with PCOS, according to the Rotterdam criteria, who are trying to conceive. Participants will be randomized to either a comprehensive lifestyle intervention program or prescribed an oral fertility medication, letrozole. These two groups will be further randomized to consume either myo-inositol or a placebo. Participants will be between the ages of 18 and 37 years. Exclusion criteria include women who have already begun fertility treatment, who are currently using myo-inositol or have taken it within the past 3 months, or who are being treated for, or have a history of, an eating disorder. The primary outcome will be the ovulation rate, the secondary outcome will be conception. Other outcomes include miscarriage rates, validated rating measures of overall quality of life (including social, relational, mind/body and emotional sub-categories) and mental health scores (depression, anxiety, and stress). DISCUSSION: This trial will determine the effectiveness of a structured lifestyle-based comprehensive intervention program for women with PCOS experiencing infertility. In addition, it will determine whether supplementing with myo-inositol provides any further benefit. The objective of this study is to assess a possible non-pharmacological solution to ovulatory dysfunction in these patients and perhaps improve other associated features of PCOS. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT02630485 . Registered on 15 December 2015.

Welsh Onion Root (Allium fistulosum) Restores Ovarian Functions from Letrozole Induced-Polycystic Ovary Syndrome.

Polycystic ovarian syndrome (PCOS) is an endocrine, metabolic, and systemic disease. It is mainly characterized by hyperandrogenism, oligomenorrhea, and high levels of luteinizing hormone (LH). There is no obvious therapy for PCOS, so patients have received symptomatic therapy. Welsh onion (Allium fistulosum) is well-known in Asian countries for its usage in food ingredients and traditional medicines. It is also studied for its many effects. These include activation of immune responses, antihypertensive effects, and antioxidant effects. Using letrozole-induced PCOS rats, we focused on herbal therapy using extract of Allium fistulosum (AF; A. fistulosum) roots to improve ovarian functions. As a nonsteroidal aromatase inhibitor, letrozole blocks conversion of testosterone to estrogen and subsequently induces PCOS phenomenon. We divided six-week-old female rats into four groups, including control, letrozole, letrozole + AF extract, and temporary letrozole groups. In our study, treatment with AF extract shows a low plasma LH/FSH ratio, and reveals high estrogen levels, ovarian morphology, folliculogenesis-related genes, and aromatase expression under PCOS mimic conditions. We concluded that AF extract administration influenced aromatase production, enhanced the estrogen steroid synthesis, and consequently restored the estrogenic feedback mechanism on the pituitary-ovary system.