Estrogen Receptor Expression Research Articles

Abstract 6286: Metabolic abnormalities are early predictors of liver metastasis in patients with ER+ breast tumors

Abstract Approximately 70% of human breast cancers express estrogen receptor alpha (ERα), providing a potential for targeted endocrine therapy for patients. Unfortunately, 30-40% of ER+ patients still experience recurrence and metastasis, with a 5-year relative overall survival rate of just 24% for patients with metastatic disease. In our preliminary analysis of the data from a large cohort of ER+ MBC patients, liver metastases were associated with reduced overall survival compared to other sites, while on the standard of care estrogen receptor antagonist, Fulvestrant (Fulv). This supports data from previous clinical trials showing that patients with liver metastases are less responsive to estrogen receptor alpha (ERα) targeting endocrine therapies compared to patients with bone or lung metastases. However, the factors underlying site-specific responses of MBC to ER antagonists are unknown. In this study, we investigated the concept that response of liver metastatic tumors is impacted by systemic glucose metabolism and the presence of liver metastatic tumors in turn will affect glucose homeostasis in the body. In our preliminary studies, metastatic liver tumors upregulated expression of enzymes that increased glycogen deposition in tumor cells in response to Fulv. Further analysis showed that a ketogenic diet restored Fulv response of metastatic liver tumors. Based on these results, we investigated if there was a relationship between metastatic site and response to endocrine therapy and if the metabolic status related to glucose homeostasis impacts metastatic disease response. We also searched if glucose homeostasis abnormalities can be indicators of liver metastasis. We obtained electronic health records from 192 patients with stage IV estrogen receptor positive breast cancer and analyzed glucose metabolism and liver panel markers including ALT, AST and ALP. Our results showed that more than 85% of patients with liver metastases have another accompanying metastasis and these patients are at a higher risk of diabetes diagnosis compared to other metastatic site patients. A higher % of patients with liver metastasis were deceased. We also observed increased levels of ALT, AST and ALP with liver metastasis and our preliminary analysis indicates that these blood biomarkers along with metabolic disease status (diabetes, hypertension, lipidemia) are good predictors of liver metastasis. Detecting early changes in markers of glucose metabolism may provide an easy diagnostic tool for early detection of liver metastases as well as assessment of treatment response. This may lead to accurate adjustment of treatment choices with improved outcome of patients with liver metastases from breast cancer. Citation Format: Ayca Nazli Mogol, Mahima Goel, Audrey Lam, Debapriya Dutta, Betsy Barnick, Maria Grosse Perdekamp, Zeynep Madak Erdogan. Metabolic abnormalities are early predictors of liver metastasis in patients with ER+ breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6286.

Abstract 1985: Ablation of adenylosuccinate lyase retarded the cell proliferation and induces mitochondrial dysfunction in endocrine therapy resistant breast cancer cells

Abstract Approximately 75% of breast tumors express estrogen receptors (ER+); recurrent ER+ breast cancer is generally incurable. Endocrine therapy (ET) is the mainstay of treatment for ER+ breast cancer and works by targeting either ER activity (Tamoxifen or Fulvestrant) or availability of its ligand estrogen (Letrozole, Exemestane). Unfortunately, endocrine therapies are limited by the emergence of resistance; resistance to endocrine therapies (ET) remains a significant clinical challenge. We have explored the role of adenylosuccinate lyase (ADSL), an enzyme of the de-novo purine biosynthesis pathway, in ET-resistant breast cancer cells. Protein expression of ADSL was significantly higher in two different ET-resistant cells (LCC9 and T47D-4HT) when compared with their respective parental ET-sensitive cells (MCF-7 and T47D). siRNA mediated ablation of ADSL in LCC9 and T47D-4HT cells blocked cell proliferation and both colony and spheroid formation. Depletion of ADSL in LCC9 cells obstructed cell cycle progression from G1 to S phase and elevated the protein expression of cyclin D1/D3, CDK2/4, and cyclin E. In T47D-4HT cells, ADSL depletion led to an accumulation of cells in S-phase with reduced protein expression of cyclin D1. Notably, ADSL ablation in LCC9 cells lead to DNA replication stress, resulting in activation (phosphorylation) of ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR) and its key downstream effector checkpoint kinase 1 (Chk1), which further limited the phosphatase activity of cdc25A leading to deactivation (phosphorylation) of Cyclin-Dependent Kinase 2 (CDK2). ADSL knockdown reduced the mitochondrial membrane potential and impaired the oxygen consumption (OCR) and extracellular acidification (ECAR) rates in both LCC9 and T47D-4HT cells. Of note, addition of AICAR to ADSL-depleted LCC9 cells significantly increased cell proliferation, colony formation, and restored mitochondrial membrane potential. RNA sequencing data revealed the effect of ADSL depletion on enrichment of different pathways in LCC9 and TH47D-4HT cells. Importantly, p53 signaling was significantly enriched in LCC9 cells but not in T47D-4HT cells after ADSL knockdown (T47D cells have a mutated p53 gene). ER+ breast cancer patients treated with ET showed high ADSL expression that is associated with poor recurrence-free survival. In summary, our findings indicate that ADSL-mediated de novo purine synthesis is crucial for cell proliferation. Therefore, targeting ADSL may be a novel therapeutic approach for ER, ET-resistant breast cancer. Citation Format: ANIL KUMAR Yadav, Lu Jin, Robert Clarke, Surojeet Sengupta. Ablation of adenylosuccinate lyase retarded the cell proliferation and induces mitochondrial dysfunction in endocrine therapy resistant breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1985.

Abstract 6279: Estrogen signaling in low-grade serous ovarian carcinoma

Abstract High level of estrogen in the blood of women has been linked to the progression and possibly development of ovarian cancer. Almost all low-grade serous ovarian cancer (LGSOC) expresses estrogen receptor alpha (ERα protein encoded by the gene ESR1) and develops during reproductive age of women. However, recent studies suggested that a higher expression of ERα correlates with better survival. In a phase II clinical trial, hormonal therapies have been shown to be associated with a clinical benefit rate of 61% for recurrent ER-positive LGSOC patients. The study also indicated that women with recurrent LGSOC who received hormonal therapies had a significantly longer progression-free survival but not overall survival. Endocrine therapies have proven to be effective in treating breast cancer. Unfortunately, in ovarian cancer clinical trials (GOG0281 and PARAGON), these treatments have not achieved comparable results, with an objective response rate of only approximately 14%. To understand the ESR1 regulation in LGSOC, we have performed RNAseq and ChIPseq analyses of LGSOC tumor tissues. We also forced over-expression of ESR1 in LGSOC cell lines. Using RNAseq data, we aligned the sequencing reads to all the known ESR1 transcript variants. We found that ESR1 transcript variant 1, 2, 3 and X11 were the major transcripts expressed. Variants 1, 2 and 3 encode the regular 66 kDa ESR1 protein, while the X11 variant encodes a 52 kDa ESR1 protein. Both 66 kDa and 52 kDa ESR1 proteins were also detected in LGSOC tumor tissues by western blots. To further explore the function of ESR1 66kDa in LGSOC, we over-expressed ESR1 variant 1 in two LGSOC cell lines (HOC-7 and HCC5075) by transfecting with an expression plasmid. Total proteins were extracted from the mock control and transfected cells for functional proteomic analysis by reverse phase protein arrays (RPPA). From the RPPA data, overexpressing ESR1 in LGSOC cell lines suppressed DNA repair pathways but activated cell cycle and mitosis pathways. To further delineate the ESR1 DNA binding sites in LGSOC, we performed ChIPseq analysis of these tumor samples as well as two ovarian cancer cell lines that express ESR1 protein. Our results demonstrated that ESR1 regulated “cistromes” in ER positive ovarian carcinoma is very different from that in ER positive breast cancer as much less ESR1 binding sites were found in LGSOC. There are ESR1 binding sites common to both types of cancers, but we found quite a few unique ESR1 binding sites. We are trying to integrate the ChIPseq and RNAseq data and hopefully identify, for the first time, an ovarian cancer specific ERα cistrome. ER expression may not always correlate with activation of ESR1 signaling pathways, which may be one of the reasons underlying endocrine therapy resistance in ovarian cancer. The results of an integrated analyses will also give us insight on the management of ER positive LGSOC by identifying new genetic targets that may be used in combination with endocrine therapy. Citation Format: Yvonne TM Tsang, Chun Wai Oscar Ng, David Gershenson, K-Kwok wong. Estrogen signaling in low-grade serous ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6279.

Abstract 4342: Breast cancer multi-omic single cell profiling identifies key progressive disease markers

Abstract Breast cancer is one of the most diagnosed cancers and has a complex tumor microenvironment (TME). Breast tumors are subtyped by receptor expression, and all have a high degree of cellular heterogeneity. We analyzed tumors from 8 individuals with early-stage breast cancer. The patients had either triple negative breast (TNBC) or an estrogen receptor (ER) positive cancer. The tumors and normal adjacent tissue were profiled by single-nuclei gene expression (GEX) and ATAC data using the 10X multi-ome methodology. Gene set analysis identified specific epithelial clusters associated with the estrogen early/late response, epithelial mesenchymal transition, and several key cytokine pathways. From those epithelial cells, a subset highly proliferative in nature emerged. It consists of both ER positive and TNBC tumor types enriched for specific DNA transcriptional motifs including those for stem cell (Yamanaka) factors and other cellular proliferation motifs. GEX proliferation markers such as MKi-67 were also highly expressed. Overlaying GEX and ATAC data, it was possible to filter the numerous differentially expressed genes and peaks into a subset of genes specific to progressive breast cancer within the proliferative cluster. Those genes included ESRP1, POP4, CCNE1, and AAMDC all of which are associated with progressive breast cancer. To further elucidate the complexity of the TME, we examined clusters of fibroblasts from our dataset. A difference in cancer associated fibroblast marker gene expression was related to the level of ER expression expressed in the tumor epithelium. In general, ER-low tumor fibroblasts had higher expression of immune cancer associated fibroblasts (iCAFs) markers while ER-positive and TNBC tumors had greater expression of extracellular matrix associated markers (emCAFs). Specific clusters were enriched for emCAFs and iCAFs representing another critical part of the TME. In conclusion, using a multi-modal single-cell approach to the TME allowed the identification of an aggressive cluster of epithelial cells marked by genes for cellular proliferation and noted therapeutic targets while also characterizing other critical TME components especially, CAFs. Citation Format: Hugh Russell, Manisha Rao, Grant Duclos, Rogelio Aguilar, Nick Barkas, Megan Callahan, Ojasvi Chaudhary, Peter Gathungu, Chris Rands, Varsha Shankarappa, Daniel Stetson, Asaf Rotem, Maurizio Scaltriti, Brian Dougherty. Breast cancer multi-omic single cell profiling identifies key progressive disease markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4342.

Linking alterations in estrogen receptor expression to memory deficits and depressive behavior in an ovariectomy mouse model

The high risk of neurological disorders in postmenopausal women is an emerging medical issue. Based on the hypothesis of altered estrogen receptors (ERα and β) after the decline of estrogen production, we investigated the changes in ERs expressions across brain regions and depressive/amnesic behaviors. C57BL/6J female mice were ovariectomized (OVX) to establish a menopausal condition. Along with behavior tests (anxiety, depression, and memory), the expression of ERs, microglial activity, and neuronal activity was measured in six brain regions (hippocampus, prefrontal cortex, striatum, raphe nucleus, amygdala, and hypothalamus) from 4 to 12 weeks after OVX. Mice exhibited anxiety- and depressive-like behaviors, as well as memory impairment. These behavioral alterations have been linked to a suppression in the expression of ERβ. The decreased ERβ expression coincided with microglial-derived neuroinflammation, as indicated by notable activations of Ionized calcium-binding adapter molecule 1 and Interleukin-1beta. Additionally, the activity of brain-derived neurotrophic factor (BDNF), particularly in the hippocampus, decreased in a time-dependent manner from 4 to 12 weeks post-OVX. Our study provides evidence shedding light on the susceptibility to memory impairment and depression in women after menopause. This susceptibility is associated with the suppression of ERβ and alteration of ERα in six brain regions.

A direct estrogenic involvement in the expression of human hypocretin

Hypocretin is synthesized exclusively in the hypothalamus and distributes inputs to several areas of the brain, which may play an important role in depression. Our previous study showed that hypocretin-1 was increased in the lateral hypothalamus in female patients with depression compared to female controls. Estrogen acts through estrogen receptor (ER)α and ERβ. We studied the possibility of a direct action of estrogen receptors on the expression of human hypocretin. We found that hypocretin-1 plasma levels were significantly higher in female patients with depression than in female controls. Female depression estrogen receptors and hypocretin are colocalized in the human lateral hypothalamus, PC12, and SK-N-SH cells. The estrogen receptor response elements (ERE) that exist in the hypocretin promoter region may directly regulate the gene expression of hypocretin. The synchronicity of change of hypocretin and estradiol both in hypothalamus and plasma was verified in female rats. In the presence of estradiol, specific binding occurs between the recombinant human ER and hypocretin-ERE. Expression of ER combined with estradiol repressed hypocretin promoter activity via the ERE. In conclusion, we found that estradiol may directly affect hypocretin neurons in the human hypothalamus via ER binding to the hypocretin-ERE, which may lead to the sex-specific pathogenesis of depression.

Is membrane androgen and estrogen receptor signaling imperative in the governing function of the adrenal cortex in the Eurasian beaver (Castor fiber L.)?

There is a need to fully know the physiology of Eurasian beaver due to its essential role in environmental homeostasis. However, a "human factor" impacts this, including stress conditions and environmental pollution. Adrenal glands protect these all. The regulation of endocrine processes by nonclassical androgen and estrogen signaling, the first and fastest control, is still a matter of research. The specific analyses performed here in mature female and male beaver adrenals contained: anatomical and histological examinations, expression and localization of membrane androgen receptor (zinc transporter, Zinc- and Iron-like protein 9; ZIP9) and membrane estrogen receptor coupled with G protein (GPER), and measurement of zinc (Zn2+) and copper (Ca2+) ion levels and corticosterone levels. We revealed normal anatomical localization, size, and tissue histology in female and male beavers, respectively. Equally,ZIP9 and GPER were localized in the membrane of all adrenal cortex cells. The protein expression of these receptors was higher (p < 0.001) in male than female adrenal cortex cells. Similarly, Zn2+ and Ca2+ ion levels were higher (p < 0.05, p < 0.01) in male than female adrenal cortex. The increased corticosterone levels (p < 0.001) were detected in the adrenal cortex of females when compared to males. The present study is the first to report the presence of nonclassical androgen and estrogen signaling and its possible regulatory function in the adrenal cortex of Eurasian beavers. We assume that this first-activated and fast-transmitted regulation can be important in the context of the effect of environmental physical and chemical stressors especially on adrenal cortex cells. The beaver adrenals may constitute an additional supplementary model for searching for universal mechanisms of adrenal cortex physiology and diseases.

G protein-coupled estrogen receptor expression in postnatal developing mouse retina.

Estrogen has emerged as a multifaceted signaling molecule in the retina, playing an important role in neural development and providing neuroprotection in adults. It interacts with two receptor types: classical estrogen receptors (ERs) alpha and beta, and G protein-coupled estrogen receptor (Gper). Gper differs from classical ERs in structure, localization, and signaling. Here we provide the first report of the temporal and spatial properties of Gper transcript and protein expression in the developing and mature mouse retina. We applied qRT-PCR to determine Gper transcript expression in wild type mouse retina from P0-P21. Immunohistochemistry and Western blot were used to determine Gper protein expression and localization at the same time points. Gper expression showed a 6-fold increase during postnatal development, peaking at P14. Relative total Gper expression exhibited a significant decrease during retinal development, although variations emerged in the timing of changes among different forms of the protein. Gper immunoreactivity was seen in retinal ganglion cells (RGCs) throughout development and also in somas in the position of horizontal cells at early time points. Immunoreactivity was observed in the cytoplasm and Golgi at all time points, in the nucleus at early time points, and in RGC axons as the retina matured. In conclusion, our study illuminates the spatial and temporal expression patterns of Gper in the developing mouse retina and provides a vital foundation for further investigations into the role of Gper in retinal development and degeneration.

Nomogram Based on US and Clinicopathologic Characteristics: Axillary Nodal Evaluation Following Neoadjuvant Chemotherapy in Patients With Node-Positive Breast Cancer

BackgroundTo develop a convenient modality to predict axillary response to neoadjuvant chemotherapy (NAC) in breast cancer patients. Materials and MethodsIn this multi-center study, a total of 1019 breast cancer patients with biopsy-proven positive lymph node (LN) receiving NAC were randomly assigned to the training and validation groups at a ratio of 7:3. Clinicopathologic and ultrasound (US) characteristics of both primary tumors and LNs were used to develop corresponding prediction models, and a nomogram integrating clinicopathologic and US predictors was generated to predict the axillary response to NAC. ResultsAxillary pathological complete response (pCR) was achieved in 47.79% of the patients. The expression of estrogen receptor, human epidermal growth factor receptor -2, Ki-67 score, and clinical nodal stage were independent predictors for nodal response to NAC. Location and radiological response of primary tumors, cortical thickness and shape of LNs on US were also significantly associated with nodal pCR. In the validation cohort, the discrimination of US model (area under the curve [AUC], 0.76) was superior to clinicopathologic model (AUC, 0.68); the combined model (AUC, 0.85) demonstrates strong discriminatory power in predicting nodal pCR. Calibration curves of the nomogram based on the combined model demonstrated that substantial agreement can be observed between the predictions and observations. This nomogram showed a false-negative rates of 16.67% in all patients and 10.53% in patients with triple negative breast cancer. ConclusionNomogram incorporating routine clinicopathologic and US characteristics can predict nodal pCR and represents a tool to aid in treatment decisions for the axilla after NAC in breast cancer patients.

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

PurposeWe 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.MethodsLevels 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.ResultsSpecific 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.ConclusionWe suggest that estrogen is essential for fetal skeletal muscle mitochondrial development and thus glucose homeostasis in adulthood.

EB1089 Increases the Antiproliferative Response of Lapatinib in Combination with Antiestrogens in HER2-Positive Breast Cancer Cells.

HER2-positive breast cancer is associated with aggressive behavior and reduced survival rates. Calcitriol restores the antiproliferative activity of antiestrogens in estrogen receptor (ER)-negative breast cancer cells by re-expressing ERα. Furthermore, calcitriol and its analog, EB1089, enhance responses to standard anti-cancer drugs. Therefore, we aimed to investigate EB1089 effects when added to the combined treatment of lapatinib and antiestrogens on the proliferation of HER2-positive breast cancer cells. BT-474 (ER-positive/HER2-positive) and SK-BR-3 (ER-negative/HER2-positive) cells were pre-treated with EB1089 to modulate ER expression. Then, cells were treated with EB1089 in the presence of lapatinib with or without the antiestrogens, and proliferation, phosphorylation array assays, and Western blot analysis were performed. The results showed that EB1089 restored the antiproliferative response to antiestrogens in SK-BR-3 cells and improved the inhibitory effects of the combination of lapatinib with antiestrogens in the two cell lines. Moreover, EB1089, alone or combined, modulated ERα protein expression and reduced Akt phosphorylation in HER2-positive cells. EB1089 significantly enhanced the cell growth inhibitory effect of lapatinib combined with antiestrogens in HER2-positive breast cancer cells by modulating ERα expression and Akt phosphorylation suppression. These results highlight the potential of this therapeutic approach as a promising strategy for managing HER2-positive breast cancer.

Sex-specific epigenetics drive low GPER expression in gastrointestinal smooth muscles in type 2 diabetic mice

Type 2 diabetes mellitus (T2D) causes gastroparesis, delayed intestinal transit, and constipation, for unknown reasons. Complications are predominant in women than men (particularly pregnant and postmenopausal women), suggesting a female hormone-mediated mechanism. Low G-protein coupled estrogen receptor (GPER) expression from epigenetic modifications may explain it. We explored sexually differentiated GPER expression and gastrointestinal symptoms related to GPER alterations in wild-type (WT) and T2D mice (db/db). We also created smooth muscle-specific GPER knockout (GPER KO) mice to phenotypically explore the effect of GPER deficiency on gastrointestinal motility. GPER mRNA and protein expression, DNA methylation and histone modifications were measured from stomach and colon samples of db/db and WT mice. Changes in gut motility were also evaluated as daily fecal pellet production patterns. We found that WT female tissues have the highest GPER mRNA and protein expressions. The expression is lowest in all db/db. GPER downregulation is associated with promoter hypermethylation and reduced enrichment of H3K4me3 and H3K27ac marks around the GPER promoter. We also observed sex-specific disparities in fecal pellet production patterns of the GPER KO mice compared to WT. We thus, conclude that T2D impairs gut GPER expression, and epigenetic sex-specific mechanisms matter in the downregulation.

Dynamic whole-body [18F]FES PET/CT increases lesion visibility in patients with metastatic breast cancer

BackgroundCorrect classification of estrogen receptor (ER) status is essential for prognosis and treatment planning in patients with breast cancer (BC). Therefore, it is recommended to sample tumor tissue from an accessible metastasis. However, ER expression can show intra- and intertumoral heterogeneity. 16α-[18F]fluoroestradiol ([18F]FES) Positron Emission Tomography/Computed Tomography (PET/CT) allows noninvasive whole-body (WB) identification of ER distribution and is usually performed as a single static image 60 min after radiotracer injection. Using dynamic whole-body (D-WB) PET imaging, we examine [18F]FES kinetics and explore whether Patlak parametric images (Ki\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${K}_{i}$$\\end{document}) are quantitative and improve lesion visibility.ResultsThis prospective study included eight patients with metastatic ER-positive BC scanned using a D-WB PET acquisition protocol. The kinetics of [18F]FES were best characterized by the irreversible two-tissue compartment model in tumor lesions and in the majority of organ tissues. Ki\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${K}_{i}$$\\end{document} values from Patlak parametric images correlated with Ki\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${K}_{i}$$\\end{document} values from the full kinetic analysis, r2 = 0.77, and with the semiquantitative mean standardized uptake value (SUVmean), r2 = 0.91. Furthermore, parametric Ki\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${K}_{i}$$\\end{document} images had the highest target-to-background ratio (TBR) in 162/164 metastatic lesions and the highest contrast-to-noise ratio (CNR) in 99/164 lesions compared to conventional SUV images. TBR was 2.45 (95% confidence interval (CI): 2.25–2.68) and CNR 1.17 (95% CI: 1.08–1.26) times higher in Ki\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${K}_{i}$$\\end{document} images compared to SUV images. These quantitative differences were seen as reduced background activity in the Ki\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${K}_{i}$$\\end{document} images.Conclusion[18F]FES uptake is best described by an irreversible two-tissue compartment model. D-WB [18F]FES PET/CT scans can be used for direct reconstruction of parametric Ki\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${K}_{i}$$\\end{document} images, with superior lesion visibility and Ki\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${K}_{i}$$\\end{document} values comparable to Ki\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${K}_{i}$$\\end{document} values found from full kinetic analyses. This may aid correct ER classification and treatment decisions.Trial registration ClinicalTrials.gov: NCT04150731, https://clinicaltrials.gov/study/NCT04150731

Premature Classification of Early-stage Endometrioid Ovarian Carcinoma With Mesonephric-like Differentiation as Mesonephric-like Adenocarcinoma.

Ovarian mesonephric-like adenocarcinoma (MLA) is a rare tumor with potential origins in endometriosis and Müllerian-type epithelial tumors. The morphologic patterns of MLA overlap with those of endometrioid ovarian carcinoma (EnOC). We speculated that a subset of MLAs would be classified as EnOCs. In this study, we attempted to identify MLAs from malignant endometrioid tumors. Given that the study patients with MLAs had both endometrioid-like and mesonephric-like morphologies, we defined mesonephric-like differentiation (MLD) as an endometrioid tumor with focal or diffuse MLA morphology and immunophenotype. Twelve patients exhibited mesonephric-like morphologic patterns. Immunohistochemistry analysis for CD10, TTF-1, estrogen receptor (ER), GATA3, calretinin, and PAX8 expression was done using whole-section slides. Two patients without the MLA immunophenotype were excluded. Ten patients with EnOCs with MLD (8.3%) were identified from a cohort of 121 patients with malignant endometrioid tumors. All 10 patients were positive for TTF-1 and/or GATA3. Most patients were ER-negative. Morphologically, MLD was associated with papillary thyroid carcinoma-like nuclei, flattened cells, tubular, nested, reticular, or glomeruloid architecture, and infiltrative growth. All 10 patients had pre-existing endometriosis and/or adenofibromas. Among the EnOCs with MLD, 5 had coexisting components such as EnOC grade 1 [(G1), cases 4, 7, and 9], mucinous borderline tumor (case 1), and dedifferentiated carcinoma (case 10), with distinct borders between EnOC with MLD and the other components. Nine of the 10 MLA patients (90%) harbored KRAS hotspot mutations. In addition, 4 patients harboring other components shared common KRAS hotspot mutations. No significant prognostic differences were observed between patients with and without MLD. Based on our findings, we suggest that EnOC with MLD, especially in the early stages and without high-grade components, should be considered a subtype of EnOC. Overtreatment should be avoided in such patients, particularly in the early stages. In this study, as the characteristics between EnOC with MLD and MLA were not distinguishable, we considered both conditions to be on the same spectrum. EnOCs with MLD exhibit the MLA phenotype during disease progression and are prematurely classified as MLA. Nevertheless, more patients with EnOC who have MLD/MLA are required for a more robust comparison between conventional EnOC according to staging and grading.

Treatment and Prognosis of Male Breast Cancer: A Multicentric, Retrospective Study Over 11 Years in the Czech Republic.

Male breast cancer (MBC) is a rare, but increasingly common disease, and lacks prospective studies. Collaborative efforts are needed to understand and address MBC, including its prognosis, in different countries. We retrospectively reviewed the clinical, histopathological, and molecular-genetic characteristics, treatments, and survival outcomes of MBC diagnosed between 2007 and 2017 in the Czech Republic. Prognostic factors of overall survival (OS), recurrence-free interval (RFi), and breast cancer-specific mortality (BCSM) were analyzed and indirectly compared to international data. We analyzed 256 patients with MBC (median age 66 years), including 12% with de novo metastatic (M1). Of 201 non-metastatic (M0) patients, 6% were <40 years old, 29% had stage I, 55% were cN0, and 54% underwent genetic testing. Overall, 97% of tumors had estrogen receptor expression ≥10%, 61% had high Ki67 index, 40% were high-grade (G3), and 68% were luminal B-like (HER2-negative). Systemic therapies included endocrine therapy (90%) and chemotherapy (53%). Few (5%) patients discontinued adjuvant endocrine therapy for reasons other than disease relapse or death. Patients treated with aromatase inhibitors alone had significantly shorter RFi (P < .001). OS, RFi, and BCSM were associated with disease stage, T stage, N stage, progesterone receptor expression, grade, and Ki67 index. Median OS reached 122 and 42 months in M0 and de novo M1 patients, respectively. Due to the rarity of MBC, this study highlights important findings from real clinical practice. Although the number of patients with MBC with unfavorable features was higher in this Czech dataset than in international studies, the prognosis remains consistent with real-world evidence.

Abstract B031: High risk no specific molecular profile (HR-NSMP) endometrial cancer can be stratified into three subgroups based on tumor grade and estrogen receptor status with differing clinicopathologic characteristics and outcomes

Abstract Introduction: Recent publications have identified ‘low risk’ (LR-NSMP) and ‘high risk’ no specific molecular profile (HR-NSMP) endometrial cancers (ECs). Although LR-NSMP (low grade (G1/2) estrogen receptor (ER) positive tumors have extremely low rates (&amp;lt;2%) of death from disease and are considered candidates for de-escalation of adjuvant therapy, little is known about the newly defined entity of HR-NSMP (G3 and/or ER-negative), including optimal management, with 27% of HR-NSMP patients dying from their disease We aimed to perform in-depth characterization of a cohort of HR-NSMP ECs to identify additional prognostic or predictive features that may inform management. Methods: Clinicopathologic data collection, immunohistochemistry (IHC), and next generation sequencing was performed on a cohort of 148 HR-NSMP ECs testing for associations with outcomes. Three subgroups of HR-NSMP were assessed: i) low grade without any ER expression (G1/2ER-, n=40), ii) high grade with ER (G3ER+, n=67), and iii) high grade without ER expression (G3ER-, n=41). Results: In a univariate analysis, advanced stage (III/IV), positive lymph node (LN) status, no LN assessment, and PIK3CA mutations were associated with inferior progression-free survival (PFS), disease specific survival (DSS), and overall survival (OS). HR-NSMP patients with positive LNs had a hazard ratio (HR) of death of 7.55 (95% CI: 3.41−16.73) compared to node negative. Additionally, patients with no LN assessment had an increased risk of death with a HR of 2.33 (95% CI: 1.12−4.85) compared to node negative. Within the three HR-NSMP subgroups, the worst OS (HR 7.06, CI 4.12-12.09, p&amp;lt; 0.001), DSS (HR 20.43, CI 10.14-41.19, p&amp;lt; 0.001), and PFS (HR 3.69, CI 1.59-8.63, p&amp;lt;0.001) were observed in patients with both adverse features (G3ER-). PIK3CA mutations were found in 32.7% of all HR-NSMP tumors tested (G1/2ER- 45.5%, G3ER+ 42.3%, G3ER- 6.7%) and associated with inferior OS (HR 4.84, CI 1.21-19.41, p=0.014), DSS (HR 4.84, CI 1.21-19.41, p=0.014), and PFS (HR 3.22, CI=1.08-9.66, p=0.028). There was a trend towards decreased OS and DSS in HR-NSMP patients with overexpression of HER2 IHC. L1CAM IHC overexpression and CTNNB1 mutation status were not associated with outcomes when assessed across all HR-NSMP or within subgroups. Conclusion: Among HR-NSMP ECs, the worst clinical outcomes were observed in patients harboring both high grade and ER negative tumors. In contrast to LR-NSMP ECs where patients who had no lymph node assessment had excellent outcomes mirroring node negative status, HR-NSMP patients who had no nodal assessment had a high rate of recurrence and death from disease suggesting the importance of comprehensive staging in this cohort. PIK3CA appears to be a prognostic stratification feature within HR-NSMP and represents a potential therapeutic pathway. More work is needed to understand the association of HER2 expression and ER status and whether HER2 -targeted therapies represent actionable opportunities for these patients. Citation Format: Andrea Neilson, Amy Jamieson, Derek Chiu, Samuel Leung, Amy Lum, Jennifer Pors, Stefan Kommoss, Aline Talhouk, David G. Huntsman, Naveena Singh, C. Blake Gilks, Jessica N. McAlpine. High risk no specific molecular profile (HR-NSMP) endometrial cancer can be stratified into three subgroups based on tumor grade and estrogen receptor status with differing clinicopathologic characteristics and outcomes [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B031.

Processed Polygonatum cyrtonema Hua attenuates postpartum depression in rat model by regulating monoamines and hormones

BackgroundPolygonatum cyrtonema Hua is a traditional Chinese medicinal food herb which can regulate the liver and Qi, nourish the heart and blood, moisten the lungs and nourish the kidneys with the potential to treat emotional diseases. However, few studies have explored the effects of Polygonatum cyrtonema Hua on postpartum depression. Therefore, we investigated whether processed Polygonatum cyrtonema Hua could improve postpartum depression in rat models by regulating monoamines and hormones. MethodsFemale Sprague-Dawley rats were randomized into normal control (0.9%Nacl), Sham operation (0.9%Nacl), postpartum depression model (0.9%Nacl), fluoxetine (2.5 mg/kg Fluoxetine), low, medium and high dose of processed Polygonatum cyrtonema Hua (2.5 g/kg, 5 g/kg, 10 g/kg) groups. Rats in these groups received drug intervention, and then subjected to Open-field test and Forced swimming test. Brain tissues and serum samples were collected and used to quantify levels of monoamines, hypothalamic-pituitary-adrenal axis and serum Estradiol. The status of neuronal cells in hippocampus 1 region was examined through hematoxylin-eosin staining, whereas expression of estrogen receptor α and β was detected by immunohistochemistry. ResultsRats in the model group showed decreased mobility time, the disorder of neuronal cells in hippocampus 1 area, and decreased concentration of 5-hydroxytryptamine and dopamine in brain tissue, norepinephrine and estradiol in serum as well as estrogen receptor α and β expression. They also exhibited increased adrenocorticotropic hormone, corticosterone and corticotropin releasing hormone in serum. However, the treatment with processed Polygonatum cyrtonem Hua or fluoxetine reversed the above abnormalities. ConclusionThe H group showed significant improvement in postpartum depression in rats, and processed Polygonatum cyrtonema Hua can be used as a developing drug for the prevention or treatment of depression.

Abstract B015: A novel Cluster 3 Endometrial Cancer PDX model to test hormone therapy regimens

Abstract Endometrial cancer is the most commonly diagnosed gynecologic cancer and continues to rise in incidence and mortality rates worldwide. With the evaluation of large datasets including the TCGA, endometrial cancers have been subdivided into four different cluster groups based on molecular characteristics. The most common subtype of endometrial cancer is the Cluster 3 category, demarcated by the expression of estrogen receptors (ER) and progesterone receptors (PR) with the potential to respond to hormonal therapy. However, Cluster 3 tumors frequently lose hormone receptor expression over time, particularly PR, making them resistant to treatment. Although numerous cell line models exist to study this disease, there is a need for improved models to select best treatment options for the specific types of endometrial cancers. Here, we describe the development of an endometrial cancer patient derived xenograft (PDX), EC-PDX3. The initial tumor from which EC-PDX3 was derived was a low-grade endometrioid endometrial carcinoma that contained a low number of somatic copy number alterations. The tumor initially tested robustly positive for both ER and PR in the donor patient as well as in initial passages in athymic mice, making this model valuable for hormone therapy research. In these studies, EC-PDX3 cells were grown within immunodeficient NSG mice. We performed H&amp;E, ER and PR staining prior to experimentation and found that while ER remained highly positive, PR levels were significantly downregulated, indicating the development of a hormone resistant state similar to that occurring in patients over time. To determine if these cells respond to hormonal therapy and if PR expression could be induced, mice were initially treated with estradiol to enhance PR expression. Next, mice were further treated with progesterone or onapristone, an experimental progestin agonist/antagonist with previously reported encouraging activity in advanced endometrial cancer in phase I and II trials. Tumors were measured weekly to determine hormone effects on cancer growth. Mice treated with estradiol alone showed substantially larger tumor growth as compared to the estradiol + progesterone and estradiol + onapristone treatment groups, indicating that hormone sensitivity was likely re-instated. RNA sequencing using Ishikawa cells in culture revealed the transcriptional effects of estradiol, progesterone and onapristone which complement the data obtained from EC-PDX3 bearing mice treated similarly. These studies identify interesting growth inhibitory effects of onapristone in endometrial cancer cells primed with estradiol including the significant induction of the cell cycle regulators such as p21 and genes involved in apoptosis. In addition, EC-PDX3 provides a model to assess hormonal therapy in Cluster 3 tumors that initially respond but develop resistance to treatment over time. Citation Format: Kimberly K. Leslie, Lane E. Smith, Jamie Padilla, Adrian Quintana. A novel Cluster 3 Endometrial Cancer PDX model to test hormone therapy regimens [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B015.

Abstract PR007: Patient derived organoids as a model to study estrogen mediated endometrial cancer

Abstract The purpose of our study was to develop a more optimal in vitro model to study the mechanisms of hormone action on endometrial cancer. Endometrial cancer (EC) represents a major health disparity with a 21% increase in mortality in African American women. The cause for this excess mortality is unknown. Endometrial cancer is commonly characterized by histological subtype into Endometrioid or Non-Endometrioid with estrogen being a driver for many endometroid type tumors. Most EC cell lines do not have high expression of estrogen receptor or do not respond to physiological levels of hormones in vitro; this lack of functional experimental models has hampered progress in the study of this disease. We have developed Patient Derived Organoids (PDOs) from 9 patient-derived xenografts (PDX) that are from both African American and Non-Hispanic Caucasian women with various stages and subtypes of EC, as a model to study EC. This model system captures the characteristics of different histological and molecular subtypes in vitro, as they maintain not only EC tumor cells but also cells from the adjacent tumor stroma. Our developed PDOs were derived from multiple molecular subtypes of EC that have different characteristics and genomic features, allowing us to observe differences in the basal properties of each tumor in vitro. The use of this organoid model system is advantageous for studying EC because growth characteristics are similar to an in vivo model, with the organoids preserving the genotype and phenotype of the native EC, allowing us to observe cellular interactions. We have observed inter-organoid variation of protein expression of β-catenin, E-cadherin, and vimentin, proteins playing a pivotal role in cellular contact, signaling, and epithelial-mesenchymal transition (EMT). Using qPCR and Western blot analysis we have observed high expression levels of estrogen receptor and progesterone receptor in endometrioid type PDOs to begin the exploration of hormonal signaling in this model. Treatment of these same PDOs with β-estradiol resulted in an increase in proliferation of the stromal cells. Single cell RNA sequencing was performed on two PDO’s that had the highest expression of progesterone and estrogen receptors. This analysis demonstrated cell population heterogeneity with multiple cell clusters forming with differential gene expression. From this analysis we can see that the cells recapitulate the gene expression from the tumor of origin, with cells containing the same genetic mutation as the original patient. These results make this model potentially good for studying hormone signaling cascades seen in EC patients that have not been previously observed in established EC cell lines. These studies will also aid in the opportunity to explore the function of differential cell types in the pathogenesis of this disease. Citation Format: Breanna M.W. Jeffcoat, Andrew B. Galdden, Russell R. Broaddus. Patient derived organoids as a model to study estrogen mediated endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr PR007.

Mapping the 5-HTergic neural pathways in perimenopausal mice and elucidating the role of oestrogen receptors in 5-HT neurotransmission

Perimenopausal syndrome (PMS) encompasses neuropsychiatric symptoms, such as hot flashes and depression, which are associated with alterations in the 5-HTergic neural pathway in the brain. However, the specific changes and mechanisms underlying these alterations remain unclear. In this study, ovariectomized mice were used to successfully establish a perimenopause model, and the changes in the expression of 5-HT and its receptors (5-HT1AR and 5-HT2AR) across 72 brain regions in these ovariectomized mice were assessed by immunohistochemistry. Although both 5-HT and 5-HT1AR were widely expressed throughout the brain, only a limited number of regions expressed 5-HT2AR. Notably, decreased expression of 5-HT was observed across almost all brain regions in the ovariectomy (OVX) group compared with the Sham group. Altered expression of both receptors was found within areas related to hot flashes (the preoptic area) or mood disorders (the amygdala). Additionally, reduced oestrogen receptor (ER)α/β expression was detected in cells in the raphe nucleus (RN), an area known to regulate body temperature. Results showed that ERα/β positively regulate the transcriptional activity of the enzymes TPH2/MAOA, which are involved in serotonin metabolism during perimenopause. This study revealed the changes in 5-HT neuropathways (5-HT, 5-HT1AR and 5-HT2AR) in perimenopausal mice, mainly in brain regions related to regulation of the body temperature, mood, sleep and memory. This study clarified that the expression of oestrogen receptor decreased in perimenopause, which regulated the transcription levels of TPH2 and MAOA, and ultimately led to the reduction of 5-HT content, providing a new target for clinical diagnosis and treatment of perimenopausal diseases.