Aromatase Inhibitor Letrozole Research Articles

Protective roles of some natural and synthetic aromatase inhibitors in testicular insufficiency caused by Bisphenol A exposure

ABSTRACT In our study, the protective role of synthetic aromatase inhibitors anastrozole (ANS), letrozole (LTZ) and exemestane (EXM) and natural aromatase inhibitors resveratrol (RSV) and apigenin (APG) against testicular failure caused by exposure to Bisphenol A (BPA) was investigated. The epididymal sperm concentration, sperm motility and sperm morphology were determined. Oxidative stress and inflammatory response parameters were examined and histological examinations were performed in testicular tissues. Our results revealed that BPA exposure decreased serum testosterone and estrogen levels, increased FSH and LH levels (p < 0.05). BPA has been found to increase oxidative stress and inflammatory response and disrupt the histological structure. Also, BPA exposure decreased testicular weight, epididymal sperm concentration and motility, and increased abnormal sperm rate (p < 0.05). These results show that ANS, LTZ and RSV treatments reduce the BPA-induced testicular damage.

Abstract PO3-23-08: Cancer and immune cells evolutionary trajectories under treatment with the aromatase inhibitor letrozole and the CDK4/6 inhibitor ribociclib

Abstract Cancer and immune cells evolutionary trajectories under treatment with the aromatase inhibitor letrozole and the CDK4/6 inhibitor ribociclib. Marie Fongaard1, Leonard Schmiester2, Salim Ghannoum1, Pål Marius Bjørnstad1, Tatjana Bosnjak3, Signe Meltzer Kleivbo3, Knut Selsås4, Stephanie Beate Geisler5, Kamilla Fjermeros5, Sameer Bhargava5, Manouchehr Seyedzadeh6, Unn-Cathrin Buvarp5, Aino Katri Rosenskiold5, Nam Thi Nguyen5, Torben Lüders7, Diether Lambrechts8, Marianne Lyngra9, Arnoldo Frigessi2, Vessela Kristensen1,10, Jürgen Geisler5,10,*, Xavier Tekpli1,* 1) Department of Medical Genetics, Oslo University Hospital, Oslo, Norway 2) University of Oslo, Oslo Centre for Biostatistics and Epidemiology (OCBE), University of Oslo, Norway 3) Novartis, Medical Affairs, Oslo, Norway 4) Department of Breast and Endocrine Surgery, Akershus University Hospital, Oslo, Norway 5) Department of Oncology, Akershus University Hospital, Oslo, Norway 6) Department of Radiology, Akershus University Hospital, Oslo, Norway 7) Department of Clinical Molecular Biology (EPIGEN), Akershus University Hospital, Oslo, Norway 8) Laboratory for Translational Genetics, Department of Human Genetics, KU Leuven, Leuven, Belgium 9) Department of Pathology, Akershus University Hospital, Oslo, Norway 10) University of Oslo, Institute of Clinical Medicine, Faculty of Medicine, Oslo, Norway&amp;lt; * Jointly supervised the work, shared corresponding authors Background The recent introduction of CDK4/6 inhibitors is regarded as a major improvement in the field of breast cancer therapy. A growing body of evidence suggests that CDK4/6 inhibitors may impact on the recruitment of immune cells within the tumor microenvironment during therapy. Further, CDK4/6 inhibitors may potentially contribute to the extinction or survival of specific cancer cell clones through evolutionary pressure exerted on breast tumors. Clinical trial The NEOLETRIB-study is a multicenter, single-arm, open-label phase II clinical trial. Patients with locally advanced, LUM-A/LUM-B breast cancer, characterized by cT3-cT4 tumors and/or cN2-3 lymph node involvement, receive ribociclib (600 mg daily, 21 days on and 7 days off) in combination with letrozole (2.5 mg daily) for a duration of 6 months in the neoadjuvant setting. Patients with large cT2 tumors are also allowed to participate. Aim of the study Using single-cell analyses at different time points of treatment, we aim at studying the evolutionary trajectories of cancer and immune cells in 30 patients participating in the NEOLETRIB-trial. Methods Tumor biopsies taken before treatment (baseline) and during treatment (day 21 of ribociclib cycles 1 and 6, respectively), underwent single-cell RNA (scRNA), T cell receptor (TCR), and B cell receptor (BCR) sequencing. The sequencing data was first processed using cellRanger and Seurat . Results Initial clustering of the scRNA enabled the identification of the main cell types in breast tumors, which included T/NK cells, B cells, epithelial cells, cancer-associated fibroblasts, myeloid cells and endothelial cells. Focusing on epithelial cells, we were distinguished cancer cells from normal epithelial cells using the InferCNV algorithm. Further, by estimating the copy number events, we determined the genetic background of clones resistant or sensitive to combination therapies for each patient, revealing the dynamics of tumor heterogeneity evolution during treatment. Additionally, we examined CD4, CD8 and NK cell types, identified different cell states within these cell types and inferred pseudotime and immune cell type differentiation trajectories. ConclusionsWe identified the cell types present in tumor biopsies from the NEOLETRIB trial. We characterized the effects of therapies on the evolution of cancer and immune cells and gain insights into the factors influencing sensitivity and resistance to the combination of letrozole and ribociclib. Citation Format: Marie Fongård, Leonard Schmiester, Salim Ghannoum, Marius Bjørnstad, Tatjana Bosnjak, Signe Kleivbo, Knut Selsås, Stephanie Geisler, Kamilla Fjermeros, Sameer Bhargava, Manouchehr Seyedzadeh, Unn-Cathrin Buvarp, Aino Vuoriluoto, Nam Thi Nguyen, Torben Lüders, Diether Lambrechts, Marianne Lyngra, Arnoldo Frigessi, Vessela Kristensen, Jürgen Geisler, Xavier Tekpli. Cancer and immune cells evolutionary trajectories under treatment with the aromatase inhibitor letrozole and the CDK4/6 inhibitor ribociclib [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-23-08.

Abstract PO4-20-03: HIPEx: Adjuvant palbociclib plus endocrine therapy in ER-positive HER2-negative early breast cancer with high recurrence score according to GenesWellTM BCT; a multi-center single arm phase II clinical trial (KCSG BR 19-13)

Abstract Background: In estrogen receptor (ER)-positive human epidermal growth factor receptor2 (HER2)-negative early breast cancer (EBC) patients, NCCN treatment guideline recommend 21-gene tests for decision of adjuvant chemotherapy if tumor size is over 0.5cm or node positive disease. In addition, GenesWellTM BCT is a prognostic assay that predict the risk of recurrence in patients with ER-positive HER2-negative and nodal stage 0-1 EBC. According to gene tests, EBC patients with high recurrence risk would have help from adjuvant chemotherapy to increase the rate of disease free survival but toxicities of chemotherapy are not negligible. Therefore, the effort for escaping adjuvant chemotherapy has been performed and currently CDK4/6 inhibitor would be considered as the substitute for cytotoxic chemotherapy in ER-positive HER2-negative EBC patients. Methods: HIPEx trial is a multi-center, phase II clinical trial to determine the efficacy of adjuvant palbociclib plus endocrine therapy in ER-positive HER2-negative EBC with high recurrence score according to GenesWellTM BCT (NCT04247633). Eligible patients are women who received curative surgery and diagnosed as ER-positive, HER2-negative and pathologic stage T1b-T2 and/or N0-1. Clinical recurrence risk is high according to the modified Adjuvant! Online guideline and GenesWellTM BCT score is 4 or more which being genomic high risk. Other criteria include ECOG performance status ≤1, adequate organ functions and availability of an archival surgical specimens. Patients receive palbociclib 125mg once daily for 21 days of 28-day cycle plus tamoxifen or aromatase inhibitor (anastrozole, letrozole or exemestane) once daily for every day. Palbociclib would be taken for two years and endocrine therapy for five years or more. If adjuvant radiotherapy is required, palbociclib should be taken two weeks after the end date of radiotherapy. If patients are premenopausal status, GnRH agonist is allowed. Primary endpoint is 3-year event free survival (EFS) rate of high clinical and genetic risk ER-positive EBC. Secondary endpoints include 3-year overall survival, quality of life, safety and tolerability of palbociclib with endocrine therapy as an adjuvant setting, exploratory analysis of predictive and prognostic biomarkers and the performance of GenesWellTM BCT for 3-year EFS rate. In total, 578 patients would be enrolled in this trial. Based on the previous results of GenesWell™ BCT in Korea, the 3-year EFS rate for the past control group (chemotherapy followed by endocrine therapy) is 87.0% and the treatment group in this clinical trial is assumed to be 90.5%.(hazard ratio for EFS = 0.717). Null hypothesis is the 3year-EFS rate in patients received adjuvant palbociclib and endocrine therapy is not less than that of the past control group. Alternative hypothesis is the 3year-EFS rate of this trial is less than that of the past control group (one-sided). Significance level is 0.025, power is 0.80. Considering 10% drop out rate, 4-years of accrual patient enrollment and 3-years of follow up period, 578 patients were required in this clinical trial. Citation Format: Ji-Yeon Kim, Sung Hoon Sim, Jieun Lee, In Hae Park, Kyong Hwa Park, Seock-Ah Im, Jee Hung Kim, Kyoung Eun Lee, Jeong Eon Lee, Jai Min Ryu, Young Kee Shin, Yeon Hee Park. HIPEx: Adjuvant palbociclib plus endocrine therapy in ER-positive HER2-negative early breast cancer with high recurrence score according to GenesWellTM BCT; a multi-center single arm phase II clinical trial (KCSG BR 19-13) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-20-03.

Quantification of the aromatase inhibitor letrozole and its carbinol metabolite in mouse plasma by UHPLC-MS/MS

A liquid chromatography - electrospray ionization-mass spectrometry (LC-ESI-MS) method was developed for the quantification of letrozole, a third-generation aromatase inhibitor, and its main carbinol metabolite (CM) in support of murine pharmacokinetic studies. Using polarity switching, simultaneous ESI-MS measurement of letrozole and CM was achieved in positive and negative mode, respectively. The assay procedure involved a one-step protein precipitation and extraction of all analytes from mouse plasma requiring only 5 μL of sample. Separation was optimized on an Accucore aQ column with gradient elution at a flow rate of 0.4 mL/min in 5 min. Two calibration curves per day over four consecutive measurement days showed satisfactory linear responses (r2 > 0.99) over concentration ranges of 5–1000 ng/mL and 20–2000 ng/mL for letrozole and CM, respectively. No matrix effect was found, and the mean extraction recoveries were 103–108 % for letrozole and 99.8–107 % for CM. Precision and accuracy within a single run and over four consecutive measurement days were verified to be within acceptable limits. Application of the developed method to preclinical pharmacokinetic studies in mice receiving oral letrozole at a dose 1 or 10 mg/kg revealed that the systemic exposure to letrozole was dose-, formulation-, and strain-dependent. These findings may inform the future design of preclinical studies aimed at refining the pharmacological profile of this clinically important drug.

Estrogen promotes fetal skeletal muscle mitochondrial distribution and ATP synthase activity important for insulin sensitivity in offspring.

We previously showed that offspring delivered to baboons in which levels of estradiol (E2) were suppressed during the second half of gestation exhibit insulin resistance. Mitochondria are essential for the production of ATP as the main source of energy for intracellular metabolic pathways, and skeletal muscle of type 2 diabetics exhibit mitochondrial abnormalities. Mitochondria express estrogen receptor β and E2 enhances mitochondrial function in adults. Therefore, the current study ascertained whether exposure of the fetus to E2 is essential for mitochondrial development. Levels of ATP synthase and citrate synthase and the morphology of mitochondria were determined in fetal skeletal muscle obtained near term from baboons untreated or treated daily with the aromatase inhibitor letrozole or letrozole plus E2. Specific activity and amount of ATP synthase were 2-fold lower (P < 0.05) in mitochondria from skeletal muscle of E2 suppressed letrozole-treated fetuses and restored to normal by treatment with letrozole plus E2. Immunocytochemistry showed that in contrast to the punctate formation of mitochondria in myocytes of untreated and letrozole plus E2 treated animals, mitochondria appeared to be diffuse in myocytes of estrogen-suppressed fetuses. However, citrate synthase activity and levels of proteins that control mitochondrial fission/fusion were similar in estrogen replete and suppressed animals. We suggest that estrogen is essential for fetal skeletal muscle mitochondrial development and thus glucose homeostasis in adulthood.

Development of an App for Symptom Management in Women With Breast Cancer Receiving Maintenance Aromatase Inhibitors: Protocol for a Mixed Methods Feasibility Study.

Patients with postmenopausal nonmetastatic estrogen receptor-positive breast cancer often experience a reduced quality of life after primary treatment. The disease and treatment trajectory consists of surgery followed by chemotherapy or radiation therapy. Upon this, maintenance hormone therapy with an aromatase inhibitor can result in several physical and psychosocial symptoms. Optimal symptom control during maintenance therapy is central to maintaining the patient's quality of life. This study aims to (1) develop an electronic symptom management tool for patients with postmenopausal early breast cancer receiving maintenance aromatase inhibitors with an endocrine aspect and (2) assess the feasibility, acceptability, and usability of the pilot version of the Bone@BC app. Furthermore, longitudinally, symptom prevalence and quality of life for patients with postmenopausal nonmetastatic estrogen receptor-positive breast cancer will be explored. This study follows a multistage research plan. In stage 1, a systematic literature review to establish an overview of aromatase inhibitor-related symptoms reported by postmenopausal women with nonmetastatic estrogen receptor-positive breast cancer will be completed. In stage 2, a comprehensive overview of symptoms related to aromatase inhibitors (letrozole, exemestane, and anastrozole) will be performed (eg, by reviewing medical leaflets and guidelines). In stage 3, an electronic app with a user-friendly Patient Concern Inventory list to comprise symptoms and concerns will be developed. Last, in stage 4, a convergent mixed methods feasibility study of the pilot version of the Bone@BC app will be conducted. A total of 45 patients with postmenopausal nonmetastatic estrogen receptor-positive breast cancer will use the app daily for symptom identification and respond to 6 serial patient-reported outcome measurements for 12 weeks. Finally, semistructured interviews will be performed. The primary outcome includes consent rate, attrition rate, retention rates, technical issues, and adherence, assessed using preestablished criteria on feasibility and a mixed methods approach for exploring acceptability. A patient advisory board consisting of 5 women with breast cancer is recruited to include their perspectives and experiences in the planning, organization, implementation, and dissemination of the research throughout the project. At the time of submitting this paper (January 2024), a total of 23 patients have been included in the stage 2 medical audit over the recruitment period of 3 months (November 2022 to February 2023), and 19 patients have been enrolled in stage 2, the semistructured patient interviews. This protocol describes a study investigating the feasibility, acceptability, and usability of the symptom management tool Bone@BC developed for patients with breast cancer with an endocrine aspect. ClinicalTrails.gov NCT05367830; https://clinicaltrials.gov/ct2/show/NCT05367830. DERR1-10.2196/49549.

An Updated Insight into Phytomolecules and Novel Approaches used in the Management of Breast Cancer.

Breast cancer is a widespread condition that kills more women from cancer-related causes than any other type of cancer globally. Women who have estrogen-dependent, initial metastatic breast cancer frequently receive treatment with surgery, radiation therapy, and chemotherapy. They may also get more specialized treatments like tamoxifen or aromatase inhibitors (anastrozole or letrozole). The World Health Organisation reported in 2012 that by 2030, breast cancer will be more common worldwide. There are several phytochemicals, such as isoflavones, coumestans, lignans, and prenylflavonoides. Isoflavones have been shown in studies to prevent the spread of breast cancer and to trigger apoptosis. Targeting BCs in metastatic breast cancer may be made possible by combining well-formulated phytochemicals in nanoparticles or other novel drug delivery agents with currently accepted endocrine and/or conventional chemotherapies. Cell signaling, regulation of cell cycles, oxidative stress action, and inflammation could be positively impacted by phytoconstituents. They have the ability to alter non-coding RNAs, to prevent the proliferation and regeneration of cancer cells. The availability of novel approaches helps in disease targeting, safety, effectiveness and efficacy. The current literature helps to know the available drugs i.e. phytoconstituents or novel drug delivery like nanoparticle, microsphere, micelles, liposomes and neosomes. The literature has been taken from PubMed, Google Scholar, SciFinder, or other internet sites.

Molecular Modeling Study of Bis-sulfonamide Derivatives Synthesis Targeting Aromatase Enzyme as Anticancer

Most studies indicate the important role of estrogen in the mechanism of occurrence and development of breast cancer. The importance of our research is the synthesis of bis-sulfonamide compounds that inhibit the aromatase enzyme, which is the main enzyme in the biosynthesis of estrogen. Molecular modeling of studied compounds was carried out by Molegro Virtual Docker (MVD) targeting aromatase enzyme and binding energy calculated to select the most encouraging compound. The highest binding energy among the studied compounds was -118.52 kcal/mol (compound A5) comparing with the aromatase substrate androstenedione -132.51 kcal/mol and the aromatase inhibitor letrozole -136.52 kcal/mol. Several of these compounds were synthesized in a simple way with good yields by reacting sulfonyl chloride derivatives with amino derivatives in an alkaline aqueous solution, or in a pyridine solution. The physicochemical characteristics and identification of synthesized compounds were determined by various analytical methods such as Mass spectrometry, Infrared spectroscopy and Nuclear Magnetic Resonance.

Effect of Aromatase Inhibitor Letrozole on the Placenta of Adult Albino Rats: A Histopathological, Immunohistochemical, and Biochemical Study.

Letrozole, an aromatase inhibitor, has recently been introduced as the preferred treatment option for ectopic pregnancy. To date, no study has investigated the effect of letrozole alone on placental tissue. The present study aimed to evaluate the effect of different doses of letrozole on the placenta of rats and to clarify the underlying mechanism. Sixty pregnant female rats were equally divided into three groups, namely the control group (GI), low-dose (0.5 mg/Kg/day) letrozole group (GII), which is equivalent to the human daily dose (HED) of 5 mg, and high-dose (1 mg/Kg/day) letrozole group (GIII), equivalent to the HED of 10 mg. Letrozole was administered by oral gavage daily from day 6 to 16 of gestation. Data were analyzed using a one-way analysis of variance followed by Tukey's post hoc test and Chi square test. P<0.05 was considered statistically significant. Compared to the GI and GII groups, high-dose letrozole significantly increased embryonic mortality with a high post-implantation loss rate (P<0.001) and significantly reduced the number of viable fetuses (P<0.001) and placental weight (P<0.001) of pregnant rats. Moreover, it significantly reduced placental estrogen receptor (ER) and progesterone receptor (PR) (P<0.001) and the expression of vascular endothelial growth factor (P<0.001), while increasing the apoptotic index of cleaved caspase-3 (P<0.001). Letrozole inhibited the expression of ER and PR in rat placenta. It interrupted stimulatory vascular signals causing significant apoptosis and placental vascular dysfunction. Letrozole in an equivalent human daily dose of 10 mg caused a high post-implantation loss rate without imposing severe side effects.

Self-fertilization in a gonochoristic fish yellow catfish

Sex control techniques are in great demand for aquaculture because of the huge benefits of mono-sex production. In a previous study on yellow catfish (Tachysurus fulvidraco), intersex individuals were produced by administrating 17α-methyltestosterone (MT) or aromatase inhibitor letrozole (LZ). The ovarian and testicular portions of intersexual gonads developed well and were completely separated, arousing our interest in producing self-fertilizable fish and clone-like fish populations. In this study, yellow catfish fry were treated with a combination of MT and LZ at a low dose (5 mg kg−1 MT, 300 mg kg−1 LZ) at a low water temperature (27.0 °C, mixed sex-producing temperature, LL group) and a high temperature (33.5 °C, male-producing temperature, HL group), and with a high dose (10 mg kg−1 MT, 600 mg kg−1 LZ) at a low water temperature (LH group), during 12–65 days post-hatching, the critical period of sex differentiation. Both XX and XY intersex were produced with different percentages at the same time through co-treatment with MT and LZ, with or without the help of male-producing temperature. The highest percentage of XY intersex (53% in XY, 36% in XX and XY) and XX intersex (47% in XX, 25% in XX and XY) was produced in LH and HL groups, respectively. Results of gonadal morphology, histology, and sperm dynamic analysis indicate that some XX and XY intersex individuals were capable of self-fertilization through artificial insemination. The reproductive performance of these intersex individuals was then investigated by injecting spawning agents, dissecting gonads, and artificially fertilizing eggs with their own sperm. The XX, XY, and YY genotypic individuals were produced by the self-fertilization of XY intersex individuals. The present study successfully produced high percentages of XX and XY intersex and conducted self-fertilization by mating the eggs and sperm of a single intersex individual. Self-fertilization in fish will show great potential in aquaculture genetics and breeding, for example, generating highly homozygous (or ‘‘clonal’’) genetic material, producing mono-sex populations, and identifying unknown sex determination modes. In particular, the cloning method of producing genetic materials will be greatly beneficial for maintaining advantageous traits through self-fertilization generation after generation.

THU644 Gene-by-PCOS Interactions In Cardio-metabolic Traits On A Panel Of Genetically Diverse Strains Of Mice

Abstract Disclosure: L.M. Velez: None. C. Johnson: None. I. Yoon: None. K. Aberra: None. R. Feng: None. I. Tamburini: None. N. Ujagar: None. M. Nelson: None. A. Senior: None. D. Ashbrook: None. R. Williams: None. D. Nicholas: None. M. Seldin: None. Polycystic ovary syndrome (PCOS) is one of the most common female endocrinopathies, and is frequently associated with major metabolic abnormalities such as insulin resistance, cardiovascular disease, and obesity. Despite the prevalence of PCOS, the genetic architecture and subsequent interactions with metabolic and reproductive variables remain relatively unexplored. Here, our aim was to define genetic interactions which mediate susceptibility to PCOS using a panel of genetically diverse strains of female mice. Leveraging accessibility in mice and defined environmental variables, we aimed to interrogate major metabolic traits, to gain mechanistic insights into the pathophysiologic outcomes associated with PCOS. Specifically, our PCOS model involved a subcutaneous (sc) implantation of an aromatase inhibitor (Letrozole)-containing pellet (or matched controls) for 6 weeks, thus promoting increased levels of circulating androgen levels. In sum, 23 strains of female mice under normal or PCOS conditions (n = 180) were subjected to in vivo echocardiographic analysis of heart function, glucose tolerance tests (GTT), body weight and composition, estrous cycle determination, and terminal assessment of testosterone (T) levels in serum. Globally, T levels, ejection fraction (EF), fat mass (FM), and AUC from GTT were increased in PCOS vs CTRLs; however, genetics remained a strong determinant: we observed substantial variation in physiologic responses to PCOS depending on the genetic background. For example, comparing PCOS vs CTRLs, some strains showed high vs low T levels and consequent insulin resistance (i.e. strains BXD124, BXD60, 129X1/SvJ, A/J, DBA/2J), increased FM (i.e.,BXD124, 129X1/SvJ, BXD48a, C3H/Hej) or increased EF (i.e., DBA/2J, NOD, BXD79), while other genetic backgrounds showed changes in insulin resistance (AUC) (i.e.,BXD75, BXD73, AKR/J), FM (i.e.,BXD73, BXD77, BXD125) or EF (i.e.,BXD77, BXD73b) without changes in T, or high vs low T with no change in insulin resistance (i.e.,BXD125, C57BL/6J), FM (i.e., A/J, BALBc/j, DBA/2J) or EF (i.e.,129, BXD124, BXD60). As part of this ongoing project we intend to categorize degrees of metabolic and reproductive abnormalities of the PCOS models and relate to human PCOS phenotypes, and, along with integration with –omics analysis of diverse key tissues, survey the genetics of inter-tissue signaling as a result of PCOS.In summary, we present a framework to study genetic differences in the context of PCOS and here we showed that the effect of a PCOS-model on key reproductive and metabolic traits is heavily modulated by the genetic background, thus highlighting future directions for the assessment of gene by PCOS interactions. Presentation: Thursday, June 15, 2023

Evaluation of the Relationship between Aromatase/Sirtuin1 Interaction and miRNA Expression in Human Neuroblastoma Cells.

Changes in activation/inhibition of Sirtuin-1 (SIRT1) and aromatase play an important role in a plethora of diseases. MicroRNAs (miRNAs) modulate multiple molecular pathways and affect a substantial number of physiological and pathological processes. The aim of this study was to investigate any possible interaction between aromatase and SIRT1 in SH-SY5Y cells and to see how there is a connection between this interaction and miRNA expression, if there is an interaction. In this study, cells were incubated in serum-deprived media for 6, 12, and 24 h. Aromatase and SIRT1 expressions were evaluated by Western blot. The IC50 concentration of SIRT1 activator (SRT1720), SIRT1 inhibitor (EX527), and aromatase inhibitors (letrozole and fadrozole) was determined by the XTT method. Then, CYP19A1 and SIRT1 levels were evaluated in the presence of SIRT1 siRNA or IC50 values for each activator/inhibitor. Finally, CYP19A1, SIRT1 expression and miRNA target gene were assessed with bioinformatic approaches. Aromatase and SIRT1 protein levels were significantly elevated in the cells incubated at 24 h in serum-deprived media (p ≤ 0.05). SIRT1 also positively regulated CYP19A1 in SH-SY5Y cells in media with/without FBS. Serum deprivation depending on time course caused changes in the oxidant/ antioxidant system. While oxidative stress index tended to decrease in the absence of FBS at 24 h compared to the control, it showed a significant decrease at 48 h in a serum-deprived manner (p ≤ 0.001). As a result of bioinformatics analysis, we determined 3 miRNAs that could potentially regulate SIRT1 and CYP19A1. hsa-miR-27a-3p and hsa-miR-181a-5p correlated in terms of their expressions at 24 h compared to 12 h, and there was a significant decrease in the expression of these miRNAs. On the contrary, the expression of hsa-miR-30c-5p significantly increased at 24 h compared to 12 h. Considering the results, a direct link between aromatase and SIRT1 was observed in human neuroblastoma cells. The identification of key miRNAs, hsa-miR-27a-3p, hsa-miR-30c-5p, and hsa-miR-181a-5p targeting both aromatase and SIRT1, provides an approach with novel insights on neurology-associated diseases.

The genomic landscape associated with resistance to aromatase inhibitors in breast cancer.

Aromatase inhibitors (AI) are drugs that are widely used in treating estrogen receptor (ER)-positive breast cancer patients. Drug resistance is a major obstacle to aromatase inhibition therapy. There are diverse reasons behind acquired AI resistance. This study aims at identifying the plausible cause of acquired AI resistance in patients administered with non-steroidal AIs (anastrozole and letrozole). We used genomic, transcriptomic, epigenetic, and mutation data of breast invasive carcinoma from The Cancer Genomic Atlas database. The data was then separated into sensitive and resistant sets based on patients' responsiveness to the non-steroidal AIs. A sensitive set of 150 patients and a resistant set of 172 patients were included for the study. These data were collectively analyzed to probe into the factors that might be responsible for AI resistance. We identified 17 differentially regulated genes (DEGs) among the two groups. Then, methylation, mutation, miRNA, copy number variation, and pathway analyses were performed for these DEGs. The top mutated genes (FGFR3, CDKN2A, RNF208, MAPK4, MAPK15, HSD3B1, CRYBB2, CDC20B, TP53TG5, and MAPK8IP3) were predicted. We also identified a key miRNA - hsa-mir-1264 regulating the expression of CDC20B. Pathway analysis revealed HSD3B1 to be involved in estrogen biosynthesis. This study reveals the involvement of key genes that might be associated with the development of AI resistance in ER-positive breast cancers and hence may act as a potential prognostic and diagnostic biomarker for these patients.

Safety and Efficacy of Tucatinib, Letrozole, and Palbociclib in Patients with Previously Treated HR+/HER2+ Breast Cancer.

To overcome resistance to antihormonal and HER2-targeted agents mediated by cyclin D1-CDK4/6 complex, we proposed an oral combination of the HER2 inhibitor tucatinib, aromatase inhibitor letrozole, and CDK4/6 inhibitor palbociclib (TLP combination) for treatment of HR+/HER2+ metastatic breast cancer (MBC). Phase Ib/II TLP trial (NCT03054363) enrolled patients with HR+/HER2+ MBC treated with ≥2 HER2-targeted agents. The phase Ib primary endpoint was safety of the regimen evaluated by NCI CTCAE version 4.3. The phase II primary endpoint was efficacy by median progression-free survival (mPFS). Forty-two women ages 22 to 81 years were enrolled. Patients received a median of two lines of therapy in the metastatic setting, 71.4% had visceral disease, 35.7% had CNS disease. The most common treatment-emergent adverse events (AE) of grade ≥3 were neutropenia (64.3%), leukopenia (23.8%), diarrhea (19.0%), and fatigue (14.3%). Tucatinib increased AUC10-19 hours of palbociclib 1.7-fold, requiring palbociclib dose reduction from 125 to 75 mg daily. In 40 response-evaluable patients, mPFS was 8.4 months, with similar mPFS in non-CNS and CNS cohorts (10.0 months vs. 8.2 months; P = 0.9). Overall response rate was 44.5%, median duration of response was 13.9 months, and clinical benefit rate was 70.4%; 60% of patients were on treatment for ≥6 months, 25% for ≥1 year, and 10% for ≥2 years. In the CNS cohort, 26.6% of patients remained on study for ≥1 year. TLP combination was safe and tolerable. AEs were expected and manageable with supportive therapy and dose reductions. TLP showed excellent efficacy for an all-oral chemotherapy-free regimen warranting further testing. See related commentary by Huppert and Rugo, p. 4993.

P-109 Letrozole versus hormone replacement therapy for frozen embryo transfer: A randomized clinical trial

Abstract Study question Do the outcomes of frozen embryo transfer cycles differ according to endometrial preparation (EP) with letrozole versus hormone replacement therapy (HRT)? Summary answer Letrozole seems to be as effective as HRT preparation in frozen cycles in terms of treatment outcomes. What is known already Vitrification improved the survival of vitrified-warmed embryos and increased the number of FET cycles. A common EP strategy for FET includes augmentation of the endometrium using estrogen followed by progesterone supplements. Retrospective studies have shown that using aromatase inhibitors (letrozole) in FET cycles was associated with higher clinical pregnancy and live birth rates, with a lower miscarriage rate, as compared to HRT cycles. Letrozole maintains regular feedback mechanisms, facilitating normal follicular growth, and promotes single follicle development and ovulation. Moreover, it does not have any negative effects on the endometrium and can improve endometrial receptivity. Study design, size, duration This randomized controlled trial was performed in two university-affiliated medical centers. A total of 170 patients undergoing frozen embryo transfer cycles from June 2018 to June 2021 were eligible. Participants/materials, setting, methods Patients were randomized to either letrozole (n = 69) or HRT protocol (n = 101) for EP, using a blocked randomization method. Inclusion criteria were single blastocyst transfer with normal uterine cavity. Exclusion criteria were patients older than 40 years, oocyte donation cycles, or more than 3 previous transfers without a pregnancy. The primary study outcome was percentage of clinical pregnancies, defined as clinical pregnancies/embryo transfers, excluding canceled treatments. Main results and the role of chance The characteristics of the study groups were comparable in age, BMI, gravidity, parity, previous treatment history and characteristics of the relevant fresh cycle in which the embryo was frozen. After excluding 5 cancelled treatments in the letrozole group and 2 in the HRT group, the percentage of clinical pregnancies between the letrozole and HRT arms were similar (24 (38.0%) versus 34 (34.6%), respectively, p = 0.67). There was no difference in the percentages of live births (22 (34.9%) versus 28 (28.6%), respectively, p = 0.4) or missed abortions (1 (4.1%) versus 5 (14.7%), p = 0.19). For patients who did not achieve pregnancy in the randomized treatment cycle, we retrospectively examined the outcome of the next treatment cycle with the other EP protocol, when possible. Achievement of a clinical pregnancy was the same for change in treatment for both study groups (7(50%) in the letrozole group and 9(50%) in the HRT group). Limitations, reasons for caution Limitations of this study included the heterogeneous study groups. Even though class II ovulation disorders were similar in prevalence in both groups, they included different indications for ART. Pregnancy complications were not evaluated and should be further investigated in a study with a larger sample size. Wider implications of the findings Letrozole seems to be as effective as HRT for EP in frozen cycles, with possible advantages of fewer missed abortions and pregnancy complications. Letrozole could be a suitable option for EP, as it is effective and, enables more convenient timing, versus natural FET. Trial registration number NCT03540979

Longitudinal trends in serum calcium, parathormone and bone mineral density in breast cancer survivors on aromatase inhibitor therapy: A single institutional cohort study.

e12531 Background: Aromatase inhibitors (AIs), when used as adjuvant treatment of hormone receptor positive breast cancer, have been associated with bone density loss. However, any impact on PTH and by extension calcium (Ca) homeostasis is yet to be elucidated as no study till date has investigated longitudinal trends in parathyroid hormone (PTH) while on AI therapy. We explored these to determine if long term AI therapy was associated with hyperparathyroidism. Methods: This is a retrospective study of female breast cancer patients at the Cleveland Clinic Foundation started on AIs (anastrozole, letrozole and exemestane) from January 2015 to April 2022. Included patients had both baseline and subsequent data (after starting AI therapy), on serum Ca, PTH and lumbar spine T-scores (LsTs). These values were compared using paired t-test. Results: We identified n=9657 patients on AI therapy and included n=249 with pre and post AI PTH values. Among these, n= 209 received anastrozole, n=34 received letrozole and n=6 received exemestane. Cohort median age was 68 (IQR 62-72) years and median duration of AI therapy was 3 years. While we observed a significant positive correlation between change in Ca and change in PTH (p=0.02), the differences between baseline and treatment median serum Ca and PTH were not statistically significant. There was no significant association between changes in either serum Ca or PTH and change in LsTs (p-value: 0.69 and p-value: 0.05 respectively). Notably, 4% patients had elevated PTH and Ca after AI therapy was initiated among whom n=4 had markedly elevated PTH with low urinary Ca. Three of these four patients also had a diagnosis of chronic kidney disease (CKD) with normal vitamin D levels. Two patients with elevated PTH were on biotin supplements. There was an overall downtrend in LsTs (p-value: 0.0039) with no significant difference between the three AIs (p-value 0.0613). However, in comparison to other AIs, letrozole had the least depreciation in LsTs. Conclusions: Our findings do not suggest any correlation between AI therapy and hyperparathyroidism among breast cancer survivors. No significant increase in PTH levels occurred during AI therapy and majority of patients with clinically significant PTH elevation also had CKD, which may have contributed also to elevation of PTH level and low urinary calcium excretion.[Table: see text]

Hormonal and genotoxic estrogen-androgen carcinogenesis in the NBL rat prostate: A role for aromatase.

Androgens are generally thought to cause prostate cancer, but the data from animal studies suggest that they must be aromatized to estrogen and act in concert with genotoxic estrogen metabolites. The objective of this study was to determine whether treatment with testosterone (T) combined with a nonestrogenic estrogen metabolite and a nongenotoxic estrogenic compound would all be necessary and sufficient for the induction of a high incidence of prostate cancer in the susceptible NBL rat strain. NBL rats were treated with low-dose testosterone via slow-release Silastic implants and with the marginally estrogenic genotoxic catechol estrogen 4-hydroxyestradiol (4OH-E2) and the nongenotoxic estrogen 2-fluoroestradiol (2F-E2) and in one experiment the aromatase inhibitor letrozole via custom-made slow-release pellets. Animals were euthanized 52 weeks after implantation and their pituitaries and prostate complexes weighed and fixed in formalin. Hematoxylin and eosin (H&E)-stained step sections were prepared and examined microscopically for proliferative lesions. Animals treated with 2F-E2, with or without the other compounds, had enlarged pituitaries demonstrating its estrogenicity. Animals treated with T, with or without the other compounds, had enlarged prostates consistent with its androgenicity. Rats treated with T plus 2F-E2 and 4OH-E2 developed a high incidence of prostatic cancer (89%), while, surprisingly, rats treated with T plus only 2F-E2 also had a high incidence of prostate cancer (95%) contradicting our initial hypothesis. To test whether the formation of E2 from T by aromatase could lead to estrogen genotoxicity and prostate carcinogenesis we then rats treated with T and 2F-E2 also with letrozole and found that it reduced prostate cancer incidence by about 50%. These findings indicate that long-term treatment with a nongenotoxic estrogen (2F-E2) and T as well as uninhibited prostatic aromatase activity generating genotoxic E2 are all required for induction of a high incidence of prostatic adenocarcinomas in NBL rats. These and previous data indicate that androgen receptor-mediated action, estrogen receptor mediation, and estrogen genotoxicity are all required and sufficient for hormonal carcinogenesis in the NBL rat prostate. Interference with the estrogen genotoxicity is a potential approach to prostate cancer chemoprevention.

Low-dose letrozole – an effective option for women with symptomatic adenomyosis awaiting IVF: a pilot randomized controlled trial

Research questionCan low-dose letrozole reduce dysmenorrhoea, menorrhagia and sonographic features in symptomatic women with adenomyosis awaiting IVF? DesignThis was a longitudinal randomized prospective pilot study to explore the effectiveness of low-dose letrozole and compare it with a gonadotropin releasing hormone (GnRH) agonist in reducing dysmenorrhoea, menorrhagia and sonographic features in symptomatic women with adenomyosis awaiting IVF. The women were treated for 3 months, either with the GnRH agonist goserelin 3.6 mg/month (n = 77) or the aromatase inhibitor letrozole 2.5 mg three times weekly (n = 79). Dysmenorrhoea and menorrhagia were evaluated at randomization and followed up monthly using a visual analogue score (VAS) and pictorial blood loss assessment chart (PBAC), respectively. A quantitative scoring method was used to assess the improvement of sonographic features after 3 months of treatment. ResultsBoth groups reported a marked improvement in symptoms after 3 months of treatment. In both the letrozole and GnRH agonist groups, VAS and PBAC scores decreased significantly over the 3 months (letrozole: P = 0.0001 and P = 0.0001 for VAS and PBAC, respectively; GnRH agonist: P = 0.0001 and P = 0.0001 for VAS and PBAC, respectively). Participants on letrozole had regular menstruation cycles, while most of the women who received the GnRH agonist were amenorrhoeic, with only four women reporting mild bleeding. Haemoglobin concentrations also improved after both treatments (letrozole P = 0.0001, GnRH agonist P = 0.0001). A quantitative assessment of sonographic features showed significant improvements following both treatments (diffuse adenomyosis of the myometrium: letrozole P = 0.015, GnRH agonist P = 0.039; diffuse adenomyosis of the junctional zone: letrozole P = 0.025, GnRH agonist P = 0.001). Women with adenomyoma also responded well to both therapies (letrozole P = 0.049, GnRH agonist P = 0.024), whereas the letrozole group responded comparatively better in focal adenomyosis when the outer myometrium was involved (letrozole P < 0.001, GnRH agonist P = 0.26). No noticeable side effects were observed in women receiving letrozole therapy. Additionally, letrozole therapy was found to be more cost-effective than GnRH agonist treatment. ConclusionsLow-dose letrozole treatment is a low-cost alternative to a GnRH agonist, with comparable effects in improving the symptoms and sonographic features of adenomyosis in women awaiting IVF.

Kinin B1 and B2 receptors mediate cancer pain associated with both the tumor and oncology therapy using aromatase inhibitors

Pain caused by the tumor or aromatase inhibitors (AIs) is a disabling symptom in breast cancer survivors. Their mechanisms are unclear, but pro-algesic and inflammatory mediators seem to be involved. Kinins are endogenous algogenic mediators associated with various painful conditions via B1 and B2 receptor activation, including chemotherapy-induced pain and breast cancer proliferation. We investigate the involvement of the kinin B1 and B2 receptors in metastatic breast tumor (4T1 breast cancer cells)-caused pain and in aromatase inhibitors (anastrozole or letrozole) therapy-associated pain. A protocol associating the tumor and antineoplastic therapy was also performed. Kinin receptors’ role was investigated via pharmacological antagonism, receptors protein expression, and kinin levels. Mechanical and cold allodynia and muscle strength were evaluated. AIs and breast tumor increased kinin receptors expression, and tumor also increased kinin levels. AIs caused mechanical allodynia and reduced the muscle strength of mice. Kinin B1 (DALBk) and B2 (Icatibant) receptor antagonists attenuated these effects and reduced breast tumor-induced mechanical and cold allodynia. AIs or paclitaxel enhanced breast tumor-induced mechanical hypersensitivity, while DALBk and Icatibant prevented this increase. Antagonists did not interfere with paclitaxel's cytotoxic action in vitro. Thus, kinin B1 or B2 receptors can be a potential target for treating the pain caused by metastatic breast tumor and their antineoplastic therapy.

Abstract GS5-07: Estradiol represses anti-tumoral immune response to promote progression of triple-negative breast cancer brain metastases

Abstract Background: Younger women with breast cancer have increased risk of development of brain metastases irrespective of the tumor subtype. We have shown that pre-menopausal levels of 17-β-Estradiol (E2) contributes to the promotion of brain metastases by influencing the tumor microenvironment. E2 promotes brain metastasis (BM) of estrogen receptor negative (ER¯) BC cells by inducing neuroinflammatory ER+ astrocytes in the brain niche to secrete pro-metastatic factors critical for early brain colonization. Ovarieoctomy (OVX) in combination with the aromatase inhibitor (Letrozole) prevented brain colonization of triple negative (TNBC) (ER-PR-HER2-) human xenografts (MDA231BR/NSG) and murine models (E0711/C57Bl6, 4T1/BALBc) through paracrine activation of EGFR and TRKB, pathways involved in increased invasion and early tumor initiation. Yet, the extent to which E2-depletion therapies can decrease progression of established BM in combination with current standard of care for brain metastasis remains unknown. Goal: Current standard of care (SOC) for patients with TNBC brain metastasis includes irradiation (SRS, whole brain) and immunotherapy (PD-1/PDL-1 inhibitors). The goal of this study was to assess how E2-depletion therapies affects brain immune function in the context of SOC for brain metastatic progression of TNBC. Results: To assess whether E2-depletion could decrease BM progression in a model that mimics standard of care for BM, TNBC E0771-GFP-luc cells were injected intracardially in syngeneic ovariectomized (OVX)-female C57Bl6 mice supplemented with pre-menopausal levels of E2. Seven days after injection (when cancer cells have colonized), mice received a single 15Gy dose brain irradiation and were randomized to continue receiving E2, E2 withdrawal (E2WD) or E2WD plus the aromatase-inhibitor letrozole (E2WD+LET). Brain metastatic burden significantly decreased in E2WD and E2WD+Letrozole treated mice as compared to E2-treated mice. Injection of E0711 cells in immunocompromised NSG mice or in the absence of brain irradiation abolished this effect, suggesting that E2-depletion therapies decrease BM progression through boosting radiation-induced anti-tumor immunity. Accordingly, there were no differences in BM progression in E2, E2WD or E2WD+let treated mice in a xenograft model (F2-7 TNBC cells) in NSG mice, even in the presence of brain irradiation. Immune-profiling of brains from OVX+E2, OVX and OVX+Let C57BL6 mice carrying BMs showed dynamic changes in immune populations at early and late stages of brain metastatic colonization. At early stages post brain colonization (3 days post ic injection) E2-treated mice showed a decreased fraction of CD11b+CD45Int CD206+ microglia/CNS macrophages as compared to OVX+LET-treated mice, without significant changes in the fraction of infiltrated lymphocytes, suggesting E2 represses early immunosurveillance through repression of microglia/CNS macrophage activation. At later stages of brain colonization (7 days post ic injection), E2-treated mice showed an increased fraction of proinflammatory microglia and decreased fraction of T and B cells as compared to OVX or OVX+let treated mice. While E2-depletion increased the recruitment of T cells to the brain niche, the fraction of CD279 (PD1+) brain T cells was similar among groups. Ongoing studies assess the efficacy of E2-depletion therapies in combination with brain radiation and PD-1 inhibitors to decrease metastatic burden and improve survival in preclinical models. Conclusion: Our results support the hypothesis that estradiol promotes brain metastatic progression by stimulating an immunosuppressive brain microenvironment. As such, FDA-approved E2-depletion therapies (aromatase inhibitors and selective-estrogen modulators) could be used in combination with brain irradiation and PD-1 inhibitors to promote a more effective anti-tumoral immune response. Citation Format: Diana Cittelly, Maria J. Contreras-Zarate, Karen ALvarez-Eraso, Vesna Tesic, Nicole Tsuji, Leanna Chafee, Sana Karam, D. Ryan Ormond, Peter Kabos. Estradiol represses anti-tumoral immune response to promote progression of triple-negative breast cancer brain metastases [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS5-07.