ESR1 Methylation Research Articles

Comparative responses to demethylating therapy in animal models of osteosarcoma.

Background The demethylating agent decitabine (DAC) effectively inhibits tumor growth and metastasis by targeting ESR1 methylation to restore estrogen receptor alpha (ERα) signaling and promoting cellular differentiation in models of human osteosarcoma (OSA). Whether this pathway can be targeted in canine OSA patients is unknown. Methods Canine OSA tumor samples were tested for ERα expression and ESR1 promoter methylation. Human (MG63.3) and canine (MC-KOS) OSA cell lines and murine xenografts were treated with DAC in vitro and in vivo , respectively. Samples were assessed using mRNA sequencing and tissue immunohistochemistry. Results ESR1 is methylated in a subset of canine OSA patient samples and the MC-KOS cell line. DAC treatment led to enhanced differentiation as demonstrated by increased ALPL expression, and suppressed tumor growth in vitro and in vivo . Metastatic progression was inhibited, particularly in the MG63.3 model, which expresses higher levels of DNA methyltransferases DNMT1 and 3B. DAC treatment induced significant alterations in immune response and cell cycle pathways. Conclusion DAC treatment activates ERα signaling, promotes bone differentiation, and inhibits tumor growth and metastasis in human and canine OSA. Additional DAC-altered pathways and species- or individual-specific differences in DNMT expression may also play a role in DAC treatment of OSA.

Transcriptomic profile of the porcine oviductal epithelial cells during the peri-conceptional period.

The optimal environment in the oviduct is created by adjusting its ultrastructure and secretory activity to serve the most suitable protection of gametes and to support embryo development. Through gametes/embryo's presence inside the oviduct, the oviductal transcriptomic profile may be altered, and these changes may be caused by DNA methylation. The results of the present study documented that in the epithelial cells of the ampulla and isthmus of the oviducts collected from pigs during the peri-conceptional period, the most differentially expressed genes (DEGs) were down-regulated. Identified DEGs were classified into gene ontology categories as well as annotated into different biological pathways. From evaluated DEGs, genes important for embryo development were selected and the level of DNA methylation was determined. It was documented CLDN18, MUC1, CYP19A3, SOCS1, and ESR1 methylation level have been altered. The presence of embryos in the oviduct changed the transcriptomic profile and the level of DNA methylation in the epithelial cells of ampulla and isthmus during the peri-conceptional period.

Comprehensive liquid biopsy analysis as a tool for the early detection of minimal residual disease in breast cancer

Liquid biopsy (LB) provides a unique minimally invasive tool to follow-up cancer patients over time, to detect minimal residual disease (MRD), to study metastasis-biology and mechanisms of therapy-resistance. Molecular characterization of CTCs offers additionally the potential to understand resistance to therapy and implement individualized targeted treatments which can be modified during the disease evolution and follow-up period of a patient. In this study, we present a long-term follow-up of operable breast cancer patients based on a comprehensive liquid biopsy analysis. We performed a comprehensive liquid biopsy analysis in peripheral blood of 13 patients with early-stage operable breast cancer at several time points for a period of ten years, consisting of: (a) CTC enumeration using the CellSearch system, (b) phenotypic analysis of CTCs using Immunofluorescence, (c) gene expression analysis, in EpCAM(+) CTCs for CK-19, CD24,CD44, ALDH1, and TWIST1, (d) analysis of PIK3CA and ESR1 mutations in EpCAM(+) CTCs and corresponding plasma ctDNA and (e) DNA methylation of ESR1 in CTCs. 10/13 (77%) patients were found negative for LB markers in PB during the whole follow-up period, and these patients did not relapse during the follow-up. However, 3/13(18%) patients that were positive for at least one LB marker relapsed within the follow-up period. The molecular characteristics of CTCs were highly different even for the same patient at different time points, and always increased before the clinical relapse. Our results indicate that liquid biopsy can reveal the presence of MRD at least 4 years before the appearance of clinically detectable metastatic disease demonstrating that a comprehensive liquid biopsy analysis provides highly important information for the therapeutic management of breast cancer patients.

Epigenetic Regulation of Estrogen Receptor Genes' Expressions in Adipose Tissue in the Course of Obesity.

Estrogen affects adipose tissue function. Therefore, this study aimed at assessing changes in the transcriptional activity of estrogen receptor (ER) α and β genes (ESR1 and ESR2, respectively) in the adipose tissues of obese individuals before and after weight loss and verifying whether epigenetic mechanisms were involved in this phenomenon. ESR1 and ESR2 mRNA and miRNA levels were evaluated using real-time PCR in visceral (VAT) and subcutaneous adipose tissue (SAT) of 78 obese (BMI > 40 kg/m2) and 31 normal-weight (BMI = 20–24.9 kg/m2) individuals and in 19 SAT samples from post-bariatric patients. ESR1 and ESR2 methylation status was studied using the methylation-sensitive digestion/real-time PCR method. Obesity was associated with a decrease in mRNA levels of both ERs in SAT (p < 0.0001) and ESR2 in VAT (p = 0.0001), while weight loss increased ESR transcription (p < 0.0001). Methylation levels of ESR1 and ESR2 promoters were unaffected. However, ESR1 mRNA in the AT of obese subjects correlated negatively with the expression of hsa-miR-18a-5p (rs = −0.444), hsa-miR-18b-5p (rs = −0.329), hsa-miR-22-3p (rs = −0.413), hsa-miR-100-5p (rs = −0.371), and hsa-miR-143-5p (rs = −0.289), while the expression of ESR2 in VAT correlated negatively with hsa-miR-576-5p (rs = −0.353) and in SAT with hsa-miR-495-3p (rs = −0.308). In conclusion, obesity-associated downregulation of ER mRNA levels in adipose tissue may result from miRNA interference.

ESR1 Methylation Measured in Cell-Free DNA to Evaluate Endocrine Resistance in Metastatic Breast Cancer Patients.

ESR1 methylation was proposed as mechanism for endocrine resistance in metastatic breast cancer patients. To evaluate its potential as a minimally invasive biomarker, we investigated the feasibility of measuring ESR1 methylation in cell-free DNA (cfDNA) and its association with endocrine resistance. First, we provided evidence that demethylation in vitro restores ER expression. Subsequently, we found that ESR1 methylation in cfDNA was not enriched in endocrine-resistant versus endocrine-sensitive patients. Interestingly, we found a correlation between ESR1 methylation and age. Publicly available data confirm an age-related increase in ESR1 methylation in leukocytes, confounding the determination of the ESR1 methylation status of tumors using cfDNA.

The high methylation level of a novel 151-bp CpG island in the ESR1 gene promoter is associated with a poor breast cancer prognosis

BackgroundThe ESR1 gene suffers methylation changes in many types of cancers, including breast cancer (BC), the most frequently diagnosed cancer in women that is also present in men. Methylation at promoter A of ESR1 is the worse prognosis in terms of overall survival; thus, the early detection, prognostic, and prediction of therapy involve some methylation biomarkers.MethodsTherefore, our study aimed to examine the methylation levels at the ESR1 gene in samples from Mexican BC patients and its possible association with menopausal status.ResultsWe identified a novel 151-bp CpG island in the promoter A of the ESR1 gene. Interestingly, methylation levels at this CpG island in positive ERα tumors were approximately 50% less than negative ERα or control samples. Furthermore, methylation levels at ESR1 were associated with menopausal status. In postmenopausal patients, the methylation levels were 1.5-fold higher than in premenopausal patients. Finally, according to tumor malignancy, triple-negative cancer subtypes had higher ESR1 methylation levels than luminal/HER2+ or luminal A subtypes.ConclusionsOur findings suggest that methylation at this novel CpG island might be a promising prognosis marker

Exercise effects on DNA methylation in EVL, CDKN2A (p14, ARF), and ESR1 in colon tissue from healthy men and women

ABSTRACT Physical activity reduces risk of colon cancer by 20–30%. Aberrant methylation patterns are common epigenetic alterations in colorectal adenomas, and cancers and play a role in cancer initiation and progression. Alterations identified in normal colon tissue represent apotential ‘field cancerization’ process, where normal colon is primed for carcinogenesis. Here, we investigate methylation patterns in three genes –Ena/VASP-like (EVL), (CDKN2A (p14, ARF)), and Oestrogen Receptor-1 (ESR1)– in normal colon tissue collected at baseline and 12 months from 202 sedentary men and women, 40–75 years, enrolled in a randomized controlled trial testing an exercise intervention vs. control (http://clinicaltrials.gov/show/NCT00668161). Participants were randomized to moderate-to-vigorous intensity exercise, 60 minutes/day, 6 days/week for 12 months, or usual lifestyle. Sigmoid colon biopsies were obtained at baseline and 12-months, DNA extracted, and bisulphite converted. Droplet digital methylation-specific PCR was performed for EVL, p14ARF, and ESR1. Generalized estimating equations modification of linear regression was used to model relationships between intervention effects and gene methylation levels, adjusting for possible confounders. There were no statistically significant differences between methylation patterns at 12-months between exercisers and controls. ESR1 methylation patterns differed by sex: women −10.58% (exercisers) +11.10% (controls); men +5.54% (exercisers), −8.16% (controls) (P=0.05), adjusting for BMI and age. There were no statistically significant changes in methylation patterns in any gene stratified by change in VO2max or minutes/week of exercise. While no statistically significant differences were found in gene methylation patterns comparing exercises vs. controls, 12-month exercise effects on ESR1 methylation differed by sex, warranting further study.

A Comprehensive Molecular Analysis of in Vivo Isolated EpCAM-Positive Circulating Tumor Cells in Breast Cancer.

Circulating tumor cell (CTC) analysis is highly promising for liquid biopsy-based molecular diagnostics. We undertook a comprehensive molecular analysis of in vivo isolated CTCs in breast cancer (BrCa). In vivo isolated CTCs from 42 patients with early and 23 patients with metastatic breast cancer (MBC) were prospectively collected and analyzed for gene expression, DNA mutations, and DNA methylation before and after treatment. 19 healthy donor (HD) samples were analyzed as a control group. In identical blood draws, CTCs were enumerated using CellSearch® and characterized by direct IF staining. All 19 HD samples were negative for CK8, CK18, CK19, ERBB2, TWIST1, VEGF, ESR1, PR, and EGFR expression, while CD44, CD24, ALDH1, VIM, and CDH2 expression was normalized to B2M (reference gene). At least one gene was expressed in 23/42 (54.8%) and 8/13 (61.5%) CTCs in early BrCa before and after therapy, and in 20/23 (87.0%) and 5/7 (71.4%) MBC before and after the first cycle of therapy. PIK3CA mutations were detected in 11/42 (26.2%) and 3/13 (23.1%) in vivo isolated CTCs in early BrCa before and after therapy, and in 11/23 (47.8%) and 2/7 (28.6%) MBC, respectively. ESR1 methylation was detected in 5/32 (15.7%) and 1/10 (10.0%) CTCs in early BrCa before and after therapy, and in 3/15(20.0%) MBC before the first line of therapy. The comprehensive molecular analysis of CTC revealed a higher sensitivity in relation to CellSearch or IF staining when based on creatine kinase selection. In vivo-CTC isolation in combination with a comprehensive molecular analysis at the gene expression, DNA mutation, and DNA methylation level comprises a highly powerful approach for molecular diagnostic applications using CTCs.

Exposure to Phthalates and Alternative Plasticizers Is Associated with Methylation Changes of ESR1 and PGR in Uterine Leiomyoma: The ELENA Study

Uterine leiomyomas are estrogen-dependent benign tumors with unknown etiologies. Phthalates are endocrine-disrupting chemicals and ubiquitous in the environment; thus, it has been suggested that they play a role in the development of uterine leiomyoma. We aimed to investigate whether the pathogenesis of uterine leiomyoma is related to methylation changes in promoter regions of estrogen receptor α (ESR1) and progesterone receptor (PGR) genes in response to phthalates and alternative plasticizers exposure. Urinary concentrations of 20 phthalate metabolites and seven metabolites of di-2-ethylhexyl terephthalate (DEHTP) and di (isononyl) cyclohexane-1,2-dicarboxylate (DINCH) were measured by UHPLC-MS/MS in thirty leiomyoma patients, who provided both paired leiomyoma and myometrium tissues. Methylation levels of ESR1 and PGR were analyzed by pyrosequencing assay. A total of 12 phthalate metabolites and 5 alternative metabolites (3 DEHTP and 2 DINCH) were detected &gt;70% among study participants. The methylation of ESR1 and PGR were significantly lower in leiomyoma tissues compared to those in myometrium (18.10 ± 4.41 vs. 28.72 ± 4.95; 2.32 ± 0.81 vs. 3.27 ± 0.56, respectively). ESR1 methylation in leiomyoma was negatively associated with mono-2-carboxylmethyl-hexyl phthalate (2cx-MMHP) and mono-3-carbocyl-propyl phthalate (MCPP) after adjusting for confounding factors. However, 1-mono-2-ethyl-5-oxohexyl-benzene-1,4-dicarboxylate (5OXO-MEHTP), one of the alternatives, showed positive association with ESR1 methylation in leiomyoma. PGR methylation in leiomyoma was significantly associated with mono butyl phthalate (MnBP), but negatively associated with cyclohexane-1,2-dicarboxylate-mono-7-hydroxy-4-methyl-heptyl ester (cx-MINCH). Our results suggest that phthalates exposure may contribute to leiomyoma pathogenesis via ESR1 and PGR methylation changes.

Induction of precocious females in the protandrous barramundi (Lates calcarifer) through implants containing 17β-estradiol - effects on gonadal morphology, gene expression and DNA methylation of key sex genes

Sex control is vital for the efficient breeding of aquaculture species. Barramundi (Lates calcarifer) is a protandrous sequential hermaphrodite that naturally sex change from male to female over its lifespan. The induction of precocious female barramundi will permit breeding of males and females of the same-generation individuals in selective breeding programs, increasing the rates of genetic gain, while reducing infrastructure costs. Accordingly, the efficacy of two dosages of 17β-estradiol (E2) delivered via implants to induce the precocious female barramundi were evaluated. Six-month-old male barramundi (405 ± 50 g body weight (BW)) were given a single cholesterol-based pellet implant containing either 0 mg E2 kg−1 BW (untreated control), 4 mg E2 kg−1 BW (‘low dose’), or 8 mg E2 kg−1 BW (‘high dose’). Changes in gonadal morphology and liver condition of implanted males, along with RT-qPCR and bisulfite amplicon sequencing to quantify expression profiles and DNA methylation of key male-female sex-related genes were then examined after 9 weeks post-implantation. Results showed that at 9 weeks post-implantation, in the ‘high dose’ E2 treatment group, 78% (7/9) of fish sex-changed completely to female, signified by gonads containing oocytes (20–30 μm) and no observed residual sperm. Comparably, 44% (4/9) of fish in the ‘low dose’ E2 treatment group had sex-changed, while remaining fish showed complete testicular regression with gonads containing only undifferentiated germ cells. In the ‘high dose’ E2 treatment, upregulation of female-biased genes (cyp19a1a and foxl2) and downregulation of male-biased genes (dmrt1, cyp11b and esr1) were observed. Increased gene expression was accompanied by decreased DNA methylation in cyp19a1a, but no significant changes in DNA methylation of foxl2 or esr1 were observed. The success of artificially-induced sex change in barramundi provides an important tool that is critical to improving selective breeding of this species.

High temperature induced masculinization of zebrafish by down-regulation of sox9b and esr1 via DNA methylation

Elevated temperature could influence the sex differentiation by altering the expression of sex-related genes in fish. However, the underlying mechanisms by which the gene expression is altered remain poorly understood. Here, we aimed to explore the role of DNA methylation in sex differentiation of zebrafish (Danio rerio) in response to elevated temperature. The results showed that high temperature (33°C) exposure of fish from 20 to 30 days post fertilization (dpf), compared to normal temperature (28°C), resulted in male-biased sex ratio and decreased expression of female-related genes including cyp19a1a, sox9b and esr1. Meanwhile, the expressions of DNA methyltransferases dnmt3a1 and dnmt3a2, and the DNA methylation levels in sox9b and esr1 promoter were significantly increased by high temperature, strongly implying that DNA methylation is involved in high temperature-induced masculinization of zebrafish. Co-treatment with 5-aza-2′-deoxycytidine (a DNA methylation inhibitor) attenuated the high temperature-induced masculinizing effect, recovered the expression of esr1 and sox9b, suppressed the transcription of dnmt3a1 and dnmt3a2, and decreased the methylation of esr1 and sox9b promoter, further confirming that DNA methylation plays an important role in high temperature-induced masculinization of zebrafish. Furthermore, the methylation of sox9b promoter decreased the enrichment of transcription factor CREB (cAMP-responsive element binding proteins). Overall, these findings suggest that high temperature induce masculinization of zebrafish by down-regulation of female-related genes via DNA methylation, providing a new insight in understanding the epigenetic mechanism of thermal-mediated sex differentiation in fish.

Sleep and Methylation of Estrogen Receptor Genes, ESR1 and GPER, in Healthy Middle-Aged and Older Women: Findings from the Women 40+ Healthy Aging Study.

PurposeSleep problems in middle-aged and older women are very common and have been associated with menopause-related changes in estrogen levels. However, not all women experience sleep problems as they enter perimenopause, and epigenetic mechanisms might contribute to the differences in sleep quality within this population. In this study, we hypothesized that increased methylation of two estrogen receptor (ER) genes (ESR1 and GPER) would be associated with increased sleep problems in healthy pre-, peri-, and postmenopausal women, either directly or indirectly through the experience of vasomotor symptoms (VMS).Materials and MethodsIn 130 healthy women aged 40–73 years, we assessed DNA methylation from dried blood spots (DBS). Women rated their sleep quality using the Pittsburgh Sleep Quality Index (PSQI), and VMS using the Menopause Rating Scale (MRS).ResultsHigher percentage methylation of ESR1 was associated with increased sleep problems, mediated by VMS, even after controlling for age, menopausal status, body mass index, estradiol levels, depressive symptoms, and caffeine consumption. There was no significant association between GPER methylation and either sleep problems or VMS.ConclusionThe study findings support an association between increased ESR1 methylation and sleep problems through increased VMS among healthy women aged 40–73 years.

Effect of folic acid and vitamin E on promoter DNA methylation and expression of TGF-β1, ESR-1 and CDH-1 in the uterus of STZ-induced diabetic rats

The present study is the first attempt made to investigate the effects of diabetes on expression and promoter DNA methylation of TGF-β1, ESR-1, and CDH-1 genes and also the effects of folic acid (FA) and vitamin E (Vit E) supplementations on improving diabetes mellitus. STZ-induced diabetic rats were treated with Vit E (200 mg/kg/day) and FA (25 mg/kg/day) for 8 weeks and expression and DNA methylation of TGF-β1, ESR-1, and CDH-1 genes in uterus were analysed. Data indicated that diabetes increases the expression of TGFβ-1 and ESR-1 and decreases CDH-1 expression and TGFβ-1 promoter methylation in the uterus of rats. Vit E and FA improved the negative effects of diabetes by decreasing the expression of TGFβ-1 and ESR-1 and increasing that of CDH-1 in diabetic rats. In conclusion, these findings emphasise that Vit E and FA supplementations could improve negative effects caused by diabetes on uterus function and fertility in diabetic rats.

Differential ESR1 Promoter Methylation in the Peripheral Blood-Findings from the Women 40+ Healthy Aging Study.

Background Estrogen receptor α (ERα) contributes to maintaining biological processes preserving health during aging. DNA methylation changes of ERα gene (ESR1) were established as playing a direct role in the regulation of ERα levels. In this study, we hypothesized decreased DNA methylation of ESR1 associated with postmenopause, lower estradiol (E2) levels, and increased age among healthy middle-aged and older women. Methods We assessed DNA methylation of ESR1 promoter region from dried blood spots (DBSs) and E2 from saliva samples in 130 healthy women aged 40–73 years. Results We found that postmenopause and lower E2 levels were associated with lower DNA methylation of a distal regulatory region, but not with DNA methylation of proximal promoters. Conclusion Our results indicate that decreased methylation of ESR1 cytosine-phosphate-guanine island (CpGI) shore may be associated with conditions of lower E2 in older healthy women.

ESR1-promoter-methylation status in primary breast cancer and its corresponding metastases.

The role of ESR1 methylation in breast cancer and its influence on disease progression is not yet fully understood. Healthy breast tissue usually does not show ESR1 promoter methylation, whereas the frequency of ESR1 methylation appears to increase in primary breast cancer and in metastatic disease. Although women with ER positive breast cancer have a good prognosis, some will relapse. We aimed to evaluate the methylation status of ESR1 in primary breast cancer and its corresponding metastases by a methylation-specific real-time PCR and to correlate the methylation status with clinical outcome. Women who were treated for primary and metastatic breast cancer were included in the study. Tumor DNA was isolated from paraffin embedded tissue sections. After bisulfite treatment ESR1 promoter methylation was analyzed by real time-MSP of each tissue sample. Kaplan-Meier-Curves were drawn for survival. In the group of patients with positive ESR1 promoter methylation in the primary breast carcinoma survival was lower compared to the group of patients without methylation (38.1months vs. 54.3months, n.s.). Seven out of 19 (37%) of those patients with positive ESR1 promoter methylation developed loss of ER expression in metastatic disease. None of the patients who had primary tumours that were ESR1 methylation negative developed ER expression negative metastatic disease. The results underline the importance of the ESR1 promoter methylation and its potential application as a predictive marker. To improve the clinical outcome of patients with metastatic disease, those with initially positive ESR1 methylation status should undergo a tissue biopsy already at the beginning of metastatic disease to identify those with loss of ER expression and thus resitance to anti-endocrine therapy.

Abstract 4577: ESR1 methylation: A liquid biopsy-based epigenetic assay for the follow up of patients with metastatic breast cancer receiving endocrine treatment

Abstract Introduction: Liquid biopsy provides real-time monitoring of tumor evolution and response to therapy through analysis of CTCs and plasma-ctDNA. ESR1 epigenetic silencing potentially affects response to endocrine treatment. We evaluated ESR1 methylation in CTCs and paired plasma-ctDNA. We evaluated ESR1 methylation in CTCs and paired plasma-ctDNA as a potential biomarker for response to everolimus/exemestane treatment. Materials and methods: A highly sensitive and specific real-time MSP assay for ESR1 methylation was developed and validated in: a) 65 primary breast tumors (FFPEs), b) EpCAM+ CTC-fractions (122 patients and 30 healthy donors; HD), c) plasma-ctDNA (108 patients and 30HD), d) in CTCs (CellSearch®) and in paired plasma-ctDNA for 58 BrCa patients. ESR1 methylation status was investigated in CTCs isolated from serial peripheral blood samples of 19 patients with ER+/ HER2- advanced BrCa receiving everolimus/exemestane. Results: ESR1 methylation was detected in: a) 25/65(38.5%) FFPEs, b) EpCAM+ CTC-fractions: 26/112(23.3%) patients and 1/30(3.3%) HD, c) plasma-ctDNA: 8/108(7.4%) patients and 1/30(3.3%) HD. ESR1 methylation was highly concordant in 58 paired DNA samples, isolated from CTCs (CellSearch®) and corresponding plasma. In serial peripheral blood samples of patients treated with everolimus/exemestane, ESR1 methylation was observed in 10/36(27.8%) CTC-positive samples, and was associated with lack of response to treatment (p=0.023 Fisher's Exact Test). Conclusions: We report for the first time the detection of ESR1 methylation in CTCs and a high concordance with paired plasma-ctDNA. ESR1 methylation in CTCs was associated with lack of response to everolimus/exemestane regimen. ESR1 methylation should be further evaluated as a potential liquid biopsy-based biomarker. Citation Format: Sofia Mastoraki, Areti Strati, Eleni Tzanikou, Maria Chimonidou, Eleni Politaki, Alexandra Voutsina, Amanda Psyrri, Vassilis Georgoulias, Evi S. Lianidou. ESR1 methylation: A liquid biopsy-based epigenetic assay for the follow up of patients with metastatic breast cancer receiving endocrine treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4577.

Abstract 4587: ESR1 methylation in primary tumors and paired circulating tumor DNA of patients with high-grade serous ovarian cancer

Abstract Background: The estrogen receptor is highly expressed in epithelial ovarian cancer and represents the main target for endocrine therapy. The ESR1 gene is frequently methylated in many types of gynecological malignancies and previous studies have shown an inverse correlation between ESR1 methylation and gene expression. Few studies attempted to investigate the role of ESR1 methylation in ovarian cancer so far, and the clinical significance of ESR1 methylation status is not as yet clear. Methods: The ESR1 methylation status was examined in primary tumors and corresponding circulating tumor DNA (ctDNA) samples of patients with high-grade serous ovarian cancer. For the detection of methylation we applied a novel highly specific and sensitive real-time methylation specific PCR (real-time MSP) assay. Two groups of primary tumor samples were recruited (training group, n=66 and validation group, n=61), along with the corresponding plasma samples (n=58) for the validation group. ESR1 methylation was also analyzed in a small group of 16 normal fallopian tube samples. Results: ESR1 was found methylated in both groups of primary tumor samples and in the corresponding plasma samples of the validation group. More specifically, ESR1 methylation was detected in 32/66 (48.5%) and 17/61 (27.9%) primary tumor samples of the training and the validation group, respectively, and in 23/58 (39.7%) corresponding plasma samples. A significant agreement between ESR1 methylation in primary tumors and paired ctDNA was observed in 40/56 (71.4%) samples of the validation group (P=0.004, k=0.360). Almost all normal fallopian tube samples (15/16) were found methylated. Interestingly, the presence of ESR1 methylation in the primary tumor samples of the validation group was nearly significantly correlated (P=0.057) with a better overall survival. Conclusions: We detected for the first time ESR1 methylation in ctDNA of patients with high-grade serous ovarian cancer. The agreement between ESR1 methylation in the primary tumors and paired ctDNA is statistically significant. Our results indicate a potential correlation between ESR1 methylation and better overall survival in high-grade serous ovarian cancer patients. Citation Format: Lydia Giannopoulou, Sofia Mastoraki, Areti Strati, Issam Chebouti, Kitty Pavlakis, Sabine Kasimir-Bauer, Evi Lianidou. ESR1 methylation in primary tumors and paired circulating tumor DNA of patients with high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4587.

Longitudinal effects of developmental bisphenol A, variable diet, and physical activity on age-related methylation in blood.

Research indicates that environmental factors can alter DNA methylation, but the specific effects of environmental exposures on epigenetic aging remain unclear. Here, using a mouse model of human-relevant exposures, we tested the hypothesis that early-life exposure to bisphenol A (BPA), variable diet, and/or changes in physical activity would modify rates of age-related methylation at several target regions, as measured from longitudinal blood samples (2, 4, and 10 months old). DNA methylation was quantified at two repetitive elements (LINE-1, IAP), two imprinted genes (Igf2, H19), and one non-imprinted gene (Esr1) in isogenic mice developmentally exposed to Control, Control + BPA (50 µg/kg diet), Western high-fat diet (WHFD), or Western + BPA diets. In blood samples, Esr1 DNA methylation increased significantly with age, but no other investigated loci showed significant age-related methylation. LINE-1 and IAP both showed significant negative environmental deflection by WHFD exposure (P < 0.05). Esr1also showed significant negative environmental deflection by WHFD exposure in female mice (P = 0.02), but not male mice. Physical activity had a non-significant positive effect on age-related Esr1 methylation in female blood, suggesting that it may partially abrogate the effects of WHFD on the aging epigenome. These results suggest that developmental nutritional exposures can modify age-related DNA methylation patterns at a gene related to growth and development. As such, environmental deflection of the aging epigenome may help to explain the growing prevalence of chronic diseases in human populations.

ESR1 methylation in primary tumors and paired circulating tumor DNA of patients with high-grade serous ovarian cancer

ObjectiveEstrogen receptor, coded by the ESR1 gene, is highly expressed in epithelial ovarian cancer. ESR1 gene is frequently methylated in many types of gynecological malignancies. However, only a few studies attempted to investigate the role of ESR1 methylation and its clinical significance in ovarian cancer so far. The aim of our study was to examine ESR1 methylation status in primary tumors and corresponding circulating tumor DNA of patients with high-grade serous ovarian cancer (HGSC). MethodsESR1 methylation was detected by a highly specific and sensitive real-time methylation-specific PCR assay. Two groups of HGSC samples were analyzed: group A (n = 66 primary tumors) and group B (n = 53 primary tumors and 50 corresponding plasma samples). ResultsESR1 was found methylated in both groups of primary tumors: in 32/66 (48.5%) of group A and in 15/53 (28.3%) of group B. 19/50 (38.0%) corresponding plasma samples of group B were also methylated for ESR1. A significant agreement for ESR1 methylation was observed between primary tumors and paired plasma ctDNA samples (P = 0.004). Interestingly, the presence of ESR1 methylation in primary tumor samples of group B was significantly correlated with a better overall survival (P = 0.027) and progression-free survival (P = 0.041). ConclusionsWe report for the first time the presence of ESR1 methylation in plasma ctDNA of patients with HGSC. The agreement between ESR1 methylation in primary tumors and paired ctDNA is statistically significant. Our results indicate a correlation between the presence of ESR1 methylation and a better clinical outcome in HGSC patients.

Gene-specific methylation profiles in BRCA-mutation positive and BRCA-mutation negative male breast cancers.

Male breast cancer (MBC) is a rare disease. Due to its rarity, MBC research and clinical approach are mostly based upon data derived from female breast cancer (FBC). Increasing evidence indicate that on molecular level MBC may be an heterogeneous disease different from FBC.In order to investigate whether epigenetic signatures could define molecular subgroups of MBCs, we performed promoter methylation analysis of genes involved in signal transduction and hormone signalling in BRCA1/2 mutation-positive and -negative MBCs.We examined 69 MBCs, paired blood samples, and 15 normal tissues for promoter methylation of hTERT, ESR1, RASSF1, AR, MYC and WNT1 genes.MBCs showed higher gene promoter methylation levels compared to paired blood and normal breast samples. Significantly higher RASSF1 methylation levels were observed in association with BRCA1/2 mutations, HER2 expression and high tumor grade. Significantly higher AR methylation levels were observed in BRCA1/2 wild-type cases and higher WNT1 methylation levels in PR negative cases.Overall, our results indicate that alterations in gene methylation profiles are common in MBC and that methylation pattern of tumor-associated genes may allow for the identification of MBC molecular subgroups, that could have implications in clinical management of MBC patients.