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The distribution of Endometrial Cancer (EC)-related deaths is uneven among the morphologic subtypes of EC. Serous Cancer (SC) makes 10% of all EC and accounts for 40% of EC-related deaths. We investigated expression of selected genes involved in epigenetic pathways by immunohistochemistry in a cohort of 106 EC patients and analyzed mRNA-based expression levels for the same set of genes in EC samples from The Cancer Genome Atlas (TCGA) dataset. A tissue microarray was constructed using low-grade (n = 30) and high-grade (n = 28) endometrioid, serous (n = 31) and clear cell carcinoma (n = 17) samples. Epigenetic marker levels were associated with PD-L1, ER/PgR, and MLH1 expression. Epigenetic markers were evaluated by H-score and PD-L1 expression was recorded by using Combined Positive Score. Results were correlated with disease stage and survival outcome. BRD4, KAT6a and HDAC9 levels were higher in SC compared to other histologic subtypes (p<0.001-0.038). After adjusting for multiple comparisons, DNMT3b expression was higher in SC compared to endometrioid-type but not between SC and CCC. The expression levels of BRD4 (p = 0.021) and KAT6a (p = 0.0027) were positively associated with PD-L abundance, while PgR (p = 0.029) and PD-L1 expression were negatively associated. In addition, BRD4 expression was low in specimens with loss of MLH1 expression (p = 0.02). More importantly, BRD4 abundance had a negative impact on disease outcome (p = 0.02). Transcriptionally, BRD4, KAT6a and DNMT3b expression levels were higher in SC in TCGA dataset. The median PD-L1 expression was marginally associated with BRD4, a transcriptional activator of CD274/PD-L1 (p = 0.069) and positively with KAT6a (p = 0.0095). In conclusion, the protein expression levels of epigenetic markers involved in cancer pathogenesis are increased by immunohistochemistry in SC. PD-L1 levels are associated with BRD4 and KAT6a in EC samples. A combination therapy with BRD4/PD-L1 or KAT6a/PD-L1 inhibitors might have a potential use in EC, in particular serous-type carcinoma., Competing Interests: he authors have declared that no competing interests exist.

Preoperative histopathological classification determines the primary surgical approach in endometrial carcinoma (EC) patients but has only moderate agreement between preoperative and postoperative diagnosis. The aim of the PIpelle Prospective ENDOmetrial carcinoma (PIPENDO) study is to determine whether histopathological assessment and a small panel of diagnostic biomarkers decreases discrepancies between preoperative and postoperative diagnosis in EC. Preoperative endometrial tissue of 378 included patients with EC was stained with 15 different antibodies. Clinically relevant discrepancies in grade or histological subtype between original preoperative and reviewed postoperative diagnosis were observed in 75 (20%) patients. Highest clinically relevant discrepancy was found in grade 2 ECs (20%), compared to 5% and 14% in respectively grade 1 and 3 endometrioid endometrial carcinomas (EECs). A practical two-biomarker panel with PR and p53 improved diagnostic accuracy (AUC = 0.92; 95%CI = 0.88-0.95) compared to solely morphological evaluation (AUC = 0.86). In preoperative high-grade EC, the diagnostic accuracy of histological subtype was improved by a three-immunohistochemical biomarker panel (PR, IMP3, and L1CAM) (AUC = 0.93; 95%CI = 0.88-0.98) compared to solely morphological evaluation (AUC = 0.81). In conclusion to improve correct preoperative diagnosis in EC, we recommend use of a panel of at least two easily accessible immunohistochemical biomarkers (PR and p53), only in grade 2 ECs. Overall, this will reduce clinically relevant discrepancies in tumor grade and subtype with postoperative diagnosis with 6% (from 20% to 14%). Addition of PR, IMP3, and L1CAM for histological subtyping in high-grade EECs resulted in a further decrease in discrepancies with 8% (from 20% to 12%)., (Copyright © 2021. Published by Elsevier Inc.)

Background: The Arias-Stella reaction (ASR) can mimic endometrial clear cell carcinoma (ECCC) in small biopsies, especially when drug or pregnancy history is unknown. A panel of immunohistochemical markers comprising napsin A, hepatocyte nuclear factor-1-beta (HNF-1β), estrogen and progesterone receptors (ER, PR) has been found useful in confirming a diagnosis of ECCC. However, the detailed characterization of how expression of this combination of markers in the ECCC mimics ASR has yet to be thoroughly evaluated., Design: The frequency and extent of napsin A, HNF-1β, ER, and PR expression in ASR were assessed in a large series. For napsin A, any cytoplasmic staining was considered positive while only nuclear staining was deemed to be positive for HNF-1β, ER, and PR. Immunohistochemical histoscores based on the intensity and extent of staining were calculated., Results: Forty cases were gestational and 10 were nongestational ASR. In 19 (38%), the reaction was extensive and involved >50% of the glands. A stromal decidual change was found in 31 (77.5%) of the gestational and 3 (30%) of the nongestational cases. Napsin A was positive in all gestational and 8 of 10 (80%) nongestational ASR. All ASR showed HNF-1β expression. ER expression was reduced in 37 (92.5%) and lost in 3 (7.5%) gestational ASR, and reduced in 9 (90%) and lost in 1 (10%) of nongestational ASR. None of the ASR in our series expressed PR., Conclusions: Naspin A and HNF-1β were frequently expressed in both gestational and nongestational ASR, and ER expression was usually either reduced or loss. Interpretation of these markers in small biopsies containing atypical clear cells should be made with caution.

Objectives: The aim of this study was to evaluate the immunohistochemical (IHC) expression of Ki-67, estrogen receptors α (ERsα), and progesterone receptors (PRs) in high-risk endometrial cancer patients and to assess their prognostic impact., Methods/materials: Immunohistochemical expression of Ki-67, ERsα, and PRs was evaluated in primary untreated endometrial cancer. The correlation among IHC staining and risk factors of recurrence such as age, Federation International of Gynecology and Obstetrics stage, grading, depth of invasion, and metastatic spread was assessed., Results: Eighty-two patients were available for the analysis. Mean ± SD age was 65.05 ± 10.48 years. The IHC assessment revealed a lack of ERα in 46.3% and of PR in 48.7% as well as a high Ki-67 in 31.7%. Loss of ERα and PR was associated with a significant higher rate of advanced stage of disease, a higher frequency of G3 tumors, and a myometrial invasion greater than 50%. A strong Ki-67 expression correlated with a deeper myometrial invasion. Analysis of the interrelationship between receptor immunonegativity revealed a relevant association of ERα immunolocalization with PR and with a high Ki-67 expression. The present study also showed that loss of ERα (P = 0.003), advanced Federation International of Gynecology and Obstetrics stage (P < 0.001), and high Ki-67 (P = 0.004) were independent prognostic factors of a shorter disease-free survival. Importantly, loss of ERα, loss of PR, and a high Ki-67 were correlated with a higher incidence of distant recurrence., Conclusions: A systematic immunohistochemistry should be a key step in the therapeutic algorithm and could contribute to the identification of high-risk tumors.

Objectives: Endometrial carcinoma mortality is mainly caused by recurrent disease, and various immunohistochemical markers to predict recurrences have been studied. Loss of the estrogen receptor (ER) and progesterone receptor (PR) and the presence of the L1 cell adhesion molecule (L1CAM) are promising markers, but their combined value has not been studied., Materials and Methods: Expression of ER, PR, and L1CAM was immunohistochemically determined in 293 endometrial carcinomas from 11 collaborating European Network for Individualized Treatment of Endometrial Cancer centers. Estrogen receptor, PR, or L1CAM staining was considered positive or negative when expressed by greater than or equal to 10% or less than 10% of the tumor cells, respectively. The association between these markers and clinicopathological markers, and their combined value in predicting survival were calculated, both in the entire cohort and in a selected groups of stage I endometrioid and low-risk stage I endometrioid carcinomas., Results: Estrogen receptor and PR were negative in 19% and 28% of the cases, respectively, and L1CAM was positive in 18%. All 3 were associated with advanced stage, high-grade, nonendometrioid histology, lymphovascular space invasion (LVSI), and reduced disease-free survival. Only advanced stage, loss of PR, and LVSI were associated with reduced disease-free survival in multivariate analysis. A prognostic model including these 3 markers was superior to 1 including only the 3 immunohistochemical markers, which was superior to the traditional model. In both the stage I endometrioid and the low-risk stage I endometrioid groups, only loss of PR was associated with reduced disease-free survival., Conclusions: Loss of ER and PR, and the presence of L1CAM are associated with high risk characteristics, and loss of PR is the strongest predictor of recurrent disease. Although a combination of these 3 markers is slightly superior to the traditional histological markers, a prognostic model including stage, PR expression, and LVSI is the most promising model in the identification of high risk carcinomas. In the stage I endometrioid carcinomas, PR immunohistochemistry appears to be of additional value in predicting recurrences.

Objective: Translational endometrial cancer (EC) research benefits from an in vitro experimental approach using EC cell lines. We demonstrated the steps that are required to examine estrogen-induced proliferative response, a simple yet important research question pertinent to EC, and devised a pragmatic methodological workflow for using EC cell lines in experimental models., Methods: Comprehensive review of all commercially available EC cell lines was carried out, and Ishikawa cell line was selected to study the estrogen responsiveness with HEC1A, RL95-2, and MFE280 cell lines as comparators where appropriate, examining relevant differential molecular (steroid receptors) and functional (phenotype, anchorage-independent growth, hormone responsiveness, migration, invasion, and chemosensitivity) characteristics in 2-dimensional and 3-dimensional cultures in vitro using immunocytochemistry, immunofluorescence, quantitative polymerase chain reaction, and Western blotting. In vivo tumor, formation, and chemosensitivity were also assessed in a chick chorioallantoic membrane model., Results: Short tandem repeat analysis authenticated the purchased cell lines, whereas gifted cells deviated significantly from the published profile. We demonstrate the importance of prior assessment of the suitability of each cell line for the chosen in vitro experimental technique. Prior establishment of baseline, nonenriched conditions was required to induce a proliferative response to estrogen. The chorioallantoic membrane model was a suitable in vivo multicellular animal model for EC for producing rapid and reproducible data., Conclusions: We have developed a methodological guide for EC researchers when using endometrial cell lines to answer important translational research questions (exemplified by estrogen-responsive cell proliferation) to facilitate robust data, while saving time and resources.

Endometrial carcinoma (EC) is a common malignant tumor in gynecology. Its incidence and development are closely associated with the levels of estrogenic and progesterone hormone. Extracellular signal-regulated kinase (ERK) signaling pathway abnormity is associated with a variety of tumors. This study detected estrogen receptor (ER), progesterone receptor (PR), ERK1, and ERK2 expression in EC and analyzed their correlations. A total of 40 EC patients in our hospital were selected as test group, while another 40 healthy volunteers were enrolled as control group. ER, PR, ERK1, and ERK2 expression in EC tissue, para-carcinoma tissue, and normal endometrial tissue were detected by immunohistochemistry and Western blot. The positive rate of ER, PR, ERK1, and ERK2 in the test group was 50%, 40%, 60%, and 65%, respectively, which were significantly higher than those in the control (P< 0.05). ER, PR, ERK1, and ERK2 protein expressions in EC cell were significantly higher than those in the control (P< 0.05). ERK1 and ERK2 presented positive correlation with ER and PR (P< 0.05). In conclusion, EC patients presented higher expressions of ER, PR, which were correlated with higher levels of ERK1 and ERK2, suggesting they might be involved in the pathogenesis of EC.

Objective: To assess the endometrial response rates to treatment with the levonorgestrel intrauterine device in post-menopausal women with atypical hyperplasia/endometrial intraepithelial neoplasia and grade 1 endometrioid (AH/EC) endometrial carcinoma who are not surgical candidates., Methods: Chart review was undertaken of patients with AH/EC who underwent levonorgestrel intrauterine device insertion by a gynecologic oncologist within two academic health systems between 2002 and 2013. When available, tissue blocks were evaluated with immunohistochemical staining for progesterone receptor expression., Results: A total of 41 patients received treatment for AH/EC with the levonorgestrel intrauterine device. Follow up sufficient to assess response occurred in 36 women (88%). Complete response was documented in 18 of 36 women (50%), no response in 8 patients (22%), partial response in 3 women (8%) and progression of disease in 7 patients (19%). Four of 18 patients with complete response (22%) later experienced relapse of hyperplasia or cancer. Four patients (10%) died during the study period: none had evidence of metastatic disease and 1 of the 4 woman died of perioperative complications following hysterectomy for stage I disease. Patients responding to treatment had significantly lower progesterone receptor expression on post-treatment biopsies., Conclusions: Intrauterine levonorgestrel is a viable treatment option for post-menopausal women with AH/EC who are poor candidates for standard surgical management. The response rate in this series is similar to published reports in premenopausal patients and includes cases of disease recurrence following conversion to benign endometrium., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Endometrial tumors arise from a hormonally responsive tissue. Defining subtypes by hormone receptor expression might better inform etiology and prediction of patient outcomes. We evaluated the potential role of tumor estrogen receptor (ER) and progesterone receptor (PR) expression to define endometrial cancer subtypes. Methods: We measured semi-continuous ER and PR protein expression in tissue specimens from 360 endometrial primary tumors from the Nurses' Health Study. To explore the impact of different definitions of marker positivity, we dichotomized ER and PR expression at different cut points in increments of 5% positive cells. Logistic regression was used to estimate associations between endometrial cancer risk factors, such as body mass index, with dichotomous ER or PR status. Reclassification statistics were used to assess whether adding dichotomous ER or PR status to standard prognostic factors of stage, grade, and histologic type would improve endometrial cancer-specific mortality prediction. Results: Compared with not being obese, obesity increased the odds of having an ER-positive tumor at cut points of 0% to 20% [maximum OR, 2.92; 95% confidence interval (CI), 1.34-6.33] as well as the odds of having a PR-positive tumor at cut points of 70% to 90% (maximum OR, 2.53; 95% CI, 1.36-4.68). Adding dichotomous tumor ER or PR status to the panel of standard predictors did not improve both model discrimination and calibration. Conclusions: Obesity may be associated with greater endometrial tumor expression of ER and PR. Adding either marker does not appear to improve mortality prediction beyond the standard predictors. Impact: Body mass index might explain some of the biological variation among endometrial tumors. Cancer Epidemiol Biomarkers Prev; 26(5); 727-35. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Objective: To assess the association of hormone receptor expression with outcome in high-grade endometrial carcinomas., Methods: This study included three sites participating in the Canadian High Risk Endometrial Cancer (CHREC) consortium. Sections from tissue microarrays containing cases with a diagnosis of endometrioid grade 3 (EC3) and endometrial serous carcinoma (ESC) were assessed for estrogen (ER) and progesterone receptor (PR) expression by immunohistochemistry. Expression was considered present if >1% of tumor cell nuclei were labeled. Associations with overall survival were assessed., Results: ER expression was present in 168/216 (78%) of EC3 and 124/192 (65%) of ESC. PR expression was present in 148/212 (70%) of EC3 and 83/196 (42%) of ESC. PR expression was significantly associated with favorable overall survival in EC3 and ESC (log rank, p=0.018 and p=0.0024) but ER expression was not. PR expression was significantly associated with favorable overall survival in EC3 independent of age, stage, center and lymph-vascular invasion (hazard ratio=0.457, 95% CI 0.257-0.811, p=0.0075) as well as in stage I and II ESC (hazard ratio=0.266, 95% CI 0.094-0.750, p=0.0123)., Conclusion: Our data provide support for the assessment of the PR expression status in EC3 and ESC. Future work will be required to determine how PR expression may be incorporated into management of patients with EC3 and ESC., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Endometrial cancer (EC) is a hormone-driven disease, and androgen receptor (AR) expression in high-grade EC (HGEC) and metastatic EC has not yet been described., Methods: The expression pattern and prognostic value of AR in relation to oestrogen (ERα and ERβ) and progesterone (PR) receptors, and the proliferation marker Ki67 in all EC subtypes (n = 85) were compared with that of healthy and hyperplastic endometrium, using immunohistochemisty and qPCR., Results: Compared with proliferative endometrium, postmenopausal endometrtial epithelium showed significantly higher expression of AR (P < 0.001) and ERα (P = 0.035), which persisted in hyperplastic epithelium and in low-grade EC (LGEC). High-grade EC showed a significant loss of AR (P < 0.0001), PR (P < 0.0001) and ERβ (P < 0.035) compared with LGEC, whilst maintaining weak to moderate ERα. Unlike PR, AR expression in metastatic lesions was significantly (P = 0.039) higher than that in primary tumours. Androgen receptor expression correlated with favourable clinicopathological features and a lower proliferation index. Loss of AR, with/without the loss of PR was associated with a significantly lower disease-free survival (P < 0.0001, P < 0.0001, respectively)., Conclusions: Postmenopausal endometrial epithelium acquires AR whilst preserving other steroid hormone receptors. Loss of AR, PR with retention of ERα and ERβ may promote the unrestrained growth of HGEC. Androgen receptor may therefore be a clinically relevant prognostic indicator and a potential therapeutic target in EC.

Objective: To examine hormone receptor expression levels and downstream gene activation in pre-treatment and post-treatment biopsies in a cohort of patients with endometrial pathology who were being conservatively managed with a progestin-containing intrauterine device (IUD). A molecular signature of treatment failure is proposed., Methods: A retrospective analysis of pre- and post-treatment biopsy specimens from 10 women treated with progestin-containing IUD for complex atypical hyperplasia (CAH) or grade 1 endometrioid adenocarcinoma was performed. Expression of estrogen receptor (ER), progesterone receptor (PR) and PR target genes was examined by immunohistochemistry (IHC) and quantitative RT-PCR., Results: The mean treatment duration was 14.3 months. Four CAH patients had stable disease or regressed after treatment, and four progressed to endometrioid adenocarcinoma. Both patients with an initial diagnosis of endometrioid adenocarcinoma regressed to CAH or no disease. In general, hormone receptor levels diminished post-treatment compared to pre-treatment biopsies; however, we noted unexpected higher expression of the B isoform of PR (PRB) as well as ER in those patients who progressed to frank cancer. There was a trend towards a non-nuclear cytoplasmic location of PRB in these patients. Importantly, the differentiating impact of PR signaling, as determined by the expression of the progestin-controlled tumor suppressor FOXO1, was lost in individuals who progressed on therapy., Conclusions: FOXO1 mRNA levels may serve as a biomarker for response to therapy and an indicator of PR function in patients being conservatively managed with a progestin-containing IUD., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The aim of this study was to compare the clinicopathological significance of miR-200a/miR-141 and miR-205 expression in endometrioid carcinomas (ECs) versus nonendometrioid carcinomas (NECs) and to assess their correlation with hormone receptor status. miR-200a/miR-141 and miR-205 expression in 154 endometrial cancers was determined by qRT-PCR. The status of estrogen and progesterone receptor (ER/PR) was assessed using immunohistochemistry. miR-200a/miR-141 and miR-205 increased significantly in ECs and in NECs. The expression level of miR-200a was significantly higher in NECs than in ECs (P=0.025). Furthermore, there was a trend that NECs with worse clinicopathological variables had a higher miR-200a expression, while an inverse trend existed in ECs. miR-205 upregulation occurred frequently in NECs without lymph node metastases (P=0.030), whereas such association was not present in ECs. Interestingly, In ECs, miR-200a/miR-141 upregulation occurred frequently in the hormone receptor positive subgroups than the negative subgroups (P<0.05). Similarly, the expression level of miR-205 was higher in the hormone receptor positive subgroups and the association between miR-205 and PR reached statistical significance (P=0.024). In contrast, in NECs, a negative correlation was found between miR-200a/miR-141 and ER or PR status. Meanwhile, in ECs, miR-200a upregulation correlated with prolonged survival in the ER positive subgroup (P=0.046), whereas an inverse trend existed in the ER negative subgroup. Our findings suggest that miR-200a/miR-141 and miR-205 increased significantly in ECs and in NECs. However, they might behave differently in ECs versus NECs. miR-200a/miR-141 and miR-205 might be associated with hormone receptor status in endometrial cancer and may possess prognostic impacts.

Unlabelled: Progesterone, acting through its receptor, PR (progesterone receptor), is the natural inhibitor of uterine endometrial carcinogenesis by inducing differentiation. PR is downregulated in more advanced cases of endometrial cancer, thereby limiting the effectiveness of hormonal therapy. Our objective was to understand and reverse the mechanisms underlying loss of PR expression in order to improve therapeutic outcomes. Using endometrial cancer cell lines and data from The Cancer Genome Atlas, our findings demonstrate that PR expression is downregulated at four distinct levels. In well-differentiated cancers, ligand-induced receptor activation and downregulation are intact. miRNAs mediate fine tuning of PR levels. As differentiation is lost, PR silencing is primarily at the epigenetic level. Initially, recruitment of the polycomb repressor complex 2 to the PR promoter suppresses transcription. Subsequently, DNA methylation prevents PR expression. Appropriate epigenetic modulators reverse these mechanisms. These data provide a rationale for combining epigenetic modulators with progestins as a therapeutic strategy for endometrial cancer., Significance: Traditional hormonal therapy for women with endometrial cancer can be molecularly enhanced by combining progestins with epigenetic modulators, thereby increasing progesterone receptor expression and significantly improving treatment efficacy.

Objective: To understand the epigenetic mechanism underlying the PR-B gene silencing in endometrial cancer (EC) cells, we compared the chromatin composition between transcriptionally active and silenced PR-B genes in EC cell lines and cancer tissues., Methods: Chromatin Immunoprecipitation (ChIP) assay was performed to measure MBD occupancy and histone acetylation/methylation in transcriptionally active and silenced PR-B genes. PR-B-positive/-negative, as well as epigenetic inhibitor-treated/-untreated EC cells were used as study models. Real-time polymerase chain reaction (PCR) and Western blot analysis were applied to measure the mRNA and protein levels of PR-B, MBD, and histones., Results: A close association among PR-B methylation, MBD binding and PR-B gene silencing was observed. Treatment with epigenetic inhibitors led to dynamic changes in the PR-B chromatin composition and gene expression. Increased H3/H4 acetylation and H3-K4 methylation, and decreased H3-K9 methylation were found to be associated with re-activation of silenced PR-B genes. MeCP2 knockdown resulted in a decreased MeCP2 binding to PR-B genes and an increased PR-B expression. ChIP analysis of MeCP2 binding to PR-B genes in the PR-B-positive/-negative EC samples confirmed the significant role of MeCP2 in PR-B silencing., Conclusion: PR-B gene expression is regulated by a concerted action of epigenetic factors including DNA methylation, MBD binding, and histone modifications. MeCP2 occupancy of PR-B genes plays a critical role in PR-B gene silencing. These findings enriched our knowledge of the epigenetic regulation of PR-B expression in EC, and suggested that the epigenetic re-activation of PR-B could be explored as a potential strategy to sensitize the PR-B-negative endometrial cancers to progestational therapy.

Endometrial cancer belongs to the commonest malignancy in females after breast cancer, malignant neoplasm of female genitals in Europe and North America but there is still not significant improvement as far as the curability of this neoplasm is concerned, especially its advanced forms. That is why there is need to define new factors that could be not only diagnostic but also predictve factors. In present study we analyzed the mRNA PTEN expression by quantitative real-time polymerase chain reaction (Q-PCR) in 123 women of endometrial carcinoma and 14 women of control group. Moreover we assessed oestrogen (ER) and progesterone receptors (PgR) in all cases. We defined the correlation between expression of PTEN gene and receptors and between PTEN expression and maturity grade of cancer. Neoplasm advancement grade G1 was diagnosed in 82.11% of patients (n = 101), G2 in 9.76% of patients (n = 12) and G3 in 8.13% of patients (n = 10). Presence of ER and PgR and decreased expression of PTEN gene was found in majority of patients with endometrial cancer (79.12% and 59.34% respectively) and the most numerous group was with weak expression of ER and strong expression of PgR. There was no statistically significant difference in gene expression depending on receptors expression nor maturity grade of cancer (p > 0.05). Evaluation of expression of PTEN gene may turn out to be a very useful tool aimed at qualifying patients for different therapies of endometrial cancer and at searching of new diagnostic and therapeutic methods of this cancer independently on its receptor status nor maturity grade of cancer.

Objective: Excessive oestrogenic stimulation is a well-known risk factor for the development and progression of endometrial cancer. Aromatase is the key enzyme which catalyses the conversion of androgens to oestrogens in postmenopausal women. Inhibition of aromatase may therefore be a useful strategy in the management of endometrial cancer. A pilot study was designed to assess the feasibility of a neoadjuvant model and understand the biological effects of anastrozole, an aromatase inhibitor, in the treatment of endometrial cancer., Methods: Patients with endometrial cancer who consented to participate in the study were randomised to receive anastrozole or placebo for a minimum of 14 days prior to definitive surgery. Endometrial samples were obtained before and after treatment. Immunohistochemistry was performed to ascertain the expression of oestrogen receptor alpha (ERα), progesterone receptor (PR), androgen receptor (AR), ki-67 and Bcl2 before and after treatment in glands and stroma of the endometrium., Results: A total of 16 patients were randomised to the anastrozole arm and 8 to the placebo arm (2:1 randomisation). A significant decrease in the glandular expression of ERα and AR was observed in the anastrozole arm. There was no significant change in the expression of PR or Bcl2. Expression of ki-67, a proliferation marker, also decreased significantly following treatment with anastrozole., Conclusions: Treatment with anastrozole caused a significant decrease in proliferation as demonstrated by decreased ki-67 expression. A large randomised controlled trial is warranted to fully assess the role of anastrozole in the neoadjuvant treatment of endometrial cancer., (© 2013.)

The purpose of this study is to assess whether composite or coordinate immunoexpression patterns of estrogen receptor (ER), progesterone receptor (PR), and Wilms tumor 1 (WT1) gene can significantly distinguish between endometrial serous carcinoma (ESC) and ovarian serous carcinoma (OSC). Immunohistochemical analyses were performed on whole tissue sections from 22 uterus-confined ESCs and on a tissue microarray of 140 high-grade, pan-stage OSCs, using antibodies to ER, PR, and WT-1. Estrogen receptor, PR, and WT1 expressions were present in 37%, 49%, and 81% of OSC, respectively, but these markers were also present in 18%, 27%, and 36% of ESC. The ER+/PR+/WT1+ coordinate profile was identified in 33.6% of OSC but in none of ESC (P = .0006), resulting in a calculated sensitivity and specificity of this profile for OSC of 33.6% and 100%, respectively. By contrast, the ER-/PR-/WT1- coordinate profile was identified in 41% of ESC but in only 6.4% of OSC (P = .0001), resulting in a calculated sensitivity and specificity of this profile for ESC of 50% and 94%. In summary, in the differential diagnosis between OSC and ESC, positivity for all 3 markers favors an extrauterine origin, whereas negativity for all 3 markers is supportive of an endometrial origin. The use of single markers for this purpose is not recommended, as each lacks optimal discriminatory power. Coordinate profiles, in general, have a high specificity but low sensitivity in this differential diagnosis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objectives: The aim of this study is to evaluate the activity and toxicity of fulvestrant, a pure estrogen receptor antagonist in patients with advanced or recurrent endometrial cancer, expressing estrogen and/or progesterone receptors (ER/PR)., Methods: Eligible patients with advanced or recurrent endometrial cancer not amenable to curative surgery and/or radiotherapy were treated with fulvestrant at a dose of 250 mg by IM injection every 4 weeks for at least 12 weeks. Therapy was continued until disease progression, death, intolerable side effects or end of study. Response was assessed in patients with at least one target lesion according to WHO-criteria., Results: Thirty-five patients were enrolled in this study and received at least one injection of fulvestrant (intention to treat-population, ITT). Twenty six patients received the intended 3 injections of fulvestrant (per protocol population, PP). There was no complete response but 4 partial responses (11.4% ITT) and 8 stable diseases. The median time to progression was 2.3 months (ITT). Overall survival was 13.2 months (ITT). Treatment was well tolerated., Conclusions: Fulvestrant at a dose of 250 mg IM every 4 weeks has marginal activity and good tolerability in patients with ER and/or PR positive advanced or recurrent endometrial cancer. A loading dose strategy and the use of 500 mg/4 weeks might improve the efficacy of this treatment., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: A phase II trial was performed to evaluate the efficacy and safety of gefitinib in patients with persistent/recurrent endometrial cancer., Methods: Women with histologically confirmed persistent/recurrent endometrial cancer were treated with 500mg oral gefitinib daily until progression or severe toxicity, with progression-free survival (PFS) at six months as the primary endpoint. Tumor expression of total epidermal growth factor receptor (EGFR), estrogen receptor (ER), progesterone receptor A (PRA) and B (PRB), Ki67, pEGFR and activated extracellular signal-regulated kinase (pERK) were examined pre- and post-treatment. EGFR was sequenced, and serum concentrations of soluble EGFR (sEGFR) at baseline also were examined., Results: Of 29 patients enrolled, 26 were evaluable for efficacy and toxicity. Four patients experienced PFS ≥6 months, and one had a complete response which was not associated with an EGFR mutation. The concentration of sEGFR in pretreatment serum was positively correlated with overall survival (OS), but not with responsiveness to gefitinib in this small patient cohort. Expression of tumor biomarkers was not associated with PFS or OS. Co-expression of ER with PRA in primary and recurrent tumors, and pEGFR with pERK in primary tumors was observed., Conclusions: This treatment regimen was tolerable but lacked sufficient efficacy to warrant further evaluation in this setting. The possible association between serum sEGFR concentrations and OS, and temporal changes in expression of pEGFR and pERK and the documented CR of one patient are interesting and warrant additional investigation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objective: Endometrial stromal sarcoma (ESS) is a rare uterine malignancy. The current treatment approaches yield unsatisfactory results, and potential therapeutic targets need exploration., Methods: We reviewed the electronic medical records of 74 patients with low-grade ESS who had been evaluated at the University of Texas MD Anderson Cancer Center between 1995 and 2006. Using immunohistochemistry, we tested the expression of targets in paraffin-embedded tissue samples taken from 13 of the patients., Results: Forty-seven patients (64%) had a recurrence, and 16 (22%) had died of their disease at last follow-up. The 10-year progression-free survival (PFS) rate was 43% (median PFS duration, 108months), and the overall survival (OS) rate was 85% (median OS, 288months). Patients who received hormonal therapy had an overall response rate of 27%; another 53% had stable disease, with a median time to progression of 24months. No complete response or partial response was observed among patients who received radiotherapy or chemotherapy. In the paraffin-embedded specimens we tested, c-abl was expressed universally. Expression of PDGF-α, PDGF-β, VEGF, and c-Kit was detected in 33%, 36%, 54%, and 8%, of specimens, respectively. EGFR and HER-2 were not detectable in any specimens., Conclusions: Our study suggests that ESS is a hormone-dependent malignancy, with hormonal therapy having activity in recurrent disease. Targeted therapy, specifically targeting c-abl may be a potential treatment for this disease., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: BAG-1 isomorphs are regulating proteins with antiapoptotic action in endometrium. ERa and PRA isomorphs seem to have an important role in endometrial cancer., Patients and Methods: We investigated the expression of BAG-1, ERa and PRA isomorphs in endometrioid adenocarcinoma and we correlated them with clinicopathological findings of the tumor. Fresh endometrial tissues were obtained from 33 patients with endometrial carcinoma and 191 paraffin-embedded tissues were analyzed by real-time PCR and immunochemistry for BAG-1, ER and PR., Results: BAG-1 protein is expressed in both nucleus and cytoplasm. Grade 3 tumors were considered to have the highest intensity. Only 4 out of 79 samples showed intense expression of ERa, while 37 samples out of 72 samples strongly expressed PRA., Conclusion: BAG-1 nuclear isomorph appeared more frequently in grade 2 tumors than in grade 1 and 3 tumors, and the cytoplasmatic isomorph was expressed more strongly than the nuclear one.

The possibility that dietary vitamin D(3) (VD(3)) exposure inhibits endometrial carcinogenesis in an animal model and modifies the enhanced risk of endometrial carcinoma associated with obesity was investigated. At 4 weeks of age, Pten(+/-) and wild-type mice were each divided into four treatment groups and fed AIN93G control diet, or AIN93G-based diet containing either 25,000 international units of VD(3) per kilogram of diet, 58% fat to induce obesity (high fat), or high fat and 25,000 international units of VD(3) per kilogram of diet. Mice were kept on these diets until they were sacrificed at week 28. Although VD(3) did not affect endometrial cancer risk, it inhibited obesity-induced increase in endometrial lesions. Specifically, high-fat diet increased focal glandular hyperplasia with atypia and malignant lesions from 58% in the control diet-fed Pten(+/-) mice to 78% in obese mice. Dietary VD(3) decreased the incidence of endometrial pathology in obese Pten(+/-) mice to 25% (P < 0.001). VD(3) altered the endometrial expression of 25-hydroxylase, 1α-hydroxylase, and vitamin D receptor in the wild-type and Pten(+/-) mice. Estrogen receptor-α mRNA levels were higher (P < 0.014) and progesterone receptor protein levels in the luminal epithelium were lower (P < 0.04) in the endometrium of control diet-fed Pten(+/-) than wild-type mice, but the expression of these receptors was not affected by the dietary exposures. VD(3) reversed the obesity-induced increase in osteopontin (P < 0.001) and significantly increased E-cadherin expression (P < 0.019) in the endometrium of obese Pten(+/-) mice. Our data confirm the known association between obesity and endometrial cancer risk. Dietary exposure to VD(3) inhibited the carcinogenic effect of obesity on the endometrium. This protective effect was linked to a reduction in the expression of osteopontin and increase in E-cadherin., (©2010 AACR.)

Objective: To determine if the triple negative phenotype (TNP) has prognostic significance in endometrial cancer with respect to various surgicopathologic outcomes and survival., Methods: A tissue microarray was constructed of 396 endometrial cancers from patients who underwent surgical staging at the Ohio State University Medical Center. Immunohistochemistry was used to test for estrogen receptor, progesterone receptor, and HER2 expression. Fluorescent in-situ hybridization (FISH) was also used to test for HER2 amplification. TNP negative patients served as controls. Pearson's chi-square was used to evaluate the association of the TNP with variables associated with a poor prognosis. Cox proportional hazards model was used to perform univariate and multivariate analyses. Progression free survival (PFS) and overall survival (OS) were analyzed with Kaplan-Meier curves, and the log rank test was used to compare the groups., Results: Twenty-seven percent of patients had the TNP. The TNP was associated with lymph node metastasis, myometrial invasion (>50%), high grade disease, non-endometrioid histology, and advanced staged disease (p<0.023 for lymph node metastasis and p<0.0001 for all others). The TNP was associated with a significantly worse survival, including a decreased PFS (p=0.009) and OS (p=0.01), but not in a fashion independent of other prognostic variables., Conclusions: The TNP is associated with advanced stage, high grade, and high risk histology, as well as poor survival. Continued investigation of the exploitation of this phenotype with targeted therapies is necessary., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

This study was aimed at determining whether high-grade endometrioid carcinomas (grade 3 International Federation of Gynecology and Obstetrics) might overlap, at least partially, non-endometrioid carcinomas (type II). To this end, a panel of clinical-pathological and immunohistochemical parameters was evaluated in three different populations: low-grade endometrioid carcinomas (LGECs; n = 57), high-grade endometrioid carcinomas (HGECs; n = 26), and non-endometrioid carcinomas (NECs; n = 30). Besides morphological appearance, HGECs appeared similar to LGECs in p53 immunostaining profile; features different from LGECs included a higher local aggressiveness, a higher invasion of lymph-vascular spaces, a lower expression of ERalpha and PR, and a higher proliferative index. HGECs were similar to NECs for local aggressiveness, invasion rate of lymph-vascular spaces, lymph node metastasis incidence, and proliferative index. HGECs, however, showed a lower rate of extra-nodal metastases, a lower incidence of p53 overexpression, and a higher positivity for ERalpha and PR. In conclusion, results from this study show that HGECs exhibit overlapping morphological and immunohistochemical features of both type I and type II endometrial carcinomas. Further research is needed to clarify the clinical value of this observation.

Determining the primary site of a uterine adenocarcinoma can be problematic in hysterectomy specimens due to the overlapping morphology of endocervical adenocarcinomas and endometrial carcinomas, particularly when both the corpus (usually lower uterine segment) and endocervix are involved and precursor lesions are lacking or difficult to distinguish from intramucosal spread of carcinoma from one site to the other. Both preferential extension of endocervical adenocarcinomas into the endometrium (rather than deep cervical stroma) and myometrial invasion derived from the endometrial component are rarely encountered; to our knowledge, these unusual patterns of spread have not been detailed in prior reports. Clinicopathologic features of 10 endocervical adenocarcinomas (9 pure, 1 adenosquamous) with prominent endometrial or endomyometrial involvement were evaluated. Tumors were analyzed for the presence of human papillomavirus (HPV) DNA and by immunohistochemistry for expression of p16 and hormone receptors. Six cases had limited amounts of tumor in the cervix proper, with depths of invasion no greater than 5 mm in 4 and only adenocarcinoma in situ in 2. Four cases had cervical stromal invasion of more than 5 mm but all of these had greater amounts of horizontal extension into endometrium or endomyometrium. Four tumors extended into endometrium only and 6 had myoinvasion associated with the endometrial component. Five tumors were originally diagnosed as primary endometrial carcinoma with either cervical extension or concurrent endocervical adenocarcinoma in situ. HPV DNA was detected in both the cervical and corpus components in all tumors and all exhibited diffuse/strong p16 expression and decreased or absent expression of hormone receptors. These ancillary techniques are useful for clarifying the origin of uterine adenocarcinomas when morphologic features and tumor location are equivocal. These cases illustrate that dominant uterine corpus involvement (endometrial or endomyometrial) by primary endocervical adenocarcinoma can lead to misclassification as primary endometrial adenocarcinoma with cervical extension (Fédération Internationale de Gynécologie et d'Obstétrique stage II), especially when endometrial extension of endocervical adenocarcinoma simulates complex atypical hyperplasia. A subset of misclassified endocervical adenocarcinomas may account for some HPV-positive uterine carcinomas reported as primary endometrial carcinomas.

Objective: The estrogen receptor (ER)-alpha and -beta and progesterone receptor (PR)-A and -B were determined in endometrioid endometrial cancer, and their prognostic values were assessed., Methods: Tissue microarrays were constructed from 315 endometrioid endometrial cancer patients. Receptor expression was assessed by immunostaining, and their semi-quantitatively determined expression levels were correlated to classical clinico-histopathological parameters in addition to disease free and disease specific survival., Results: Patients were classified as FIGO stage I (59.0%), stage II (17.1%), stage III (19.4%) and stage IV (4.1%). Sixty-five patients (20.6%) developed recurrent disease and 38 (12.1%) died due to endometrial cancer. In univariate analysis, expression of ER-alpha was related to early stage endometrial cancer (p=0.020), while expression of ER-alpha, PR-A and PR-B was associated with lower grade tumours (p<0.0001, p<0.001 and p=0.001 respectively). A ratio of ER-alpha/ER-beta <1 was related to a shorter disease free survival (p=0.027), while the ratio of PR-A/PR-B <1 both was associated with a shorter disease free survival as well as a shorter overall survival (p=0.044 and p=0.005, respectively). In early stage disease, using multivariate analysis, absence of ER-alpha was independently related to death of disease (p=0.017, OR 7.28, 95% CI 1.42-37.25), while absence of PR-A (p=0.015, OR 4.2, 95% CI 1.32-13.33) appeared to be an independent prognostic factor for relapse of disease., Conclusion: We conclude that in early stage endometrioid endometrial cancer absence of PR-A is an independent prognostic factor for disease-free survival, while patients with ER-alpha positive tumours have a better overall survival.

Introduction: The goal of this study was to explore the relationship between the expression of hormone receptors in metastatic endometrial tumors and clinical response to daily tamoxifen citrate and intermittent weekly medroxyprogesterone acetate., Study Design: Patients with measurable recurrent or advanced endometrial cancer were enrolled on a clinical trial, Gynecologic Oncology Group Study 119. A pretreatment tumor biopsy was obtained and subjected to immunohistochemical analyses. Estrogen receptor-alpha (ER-alpha) and progesterone receptor (PR) were assessed on frozen tissues, and PR isoforms A and B were detected on fixed tissues. The receptors were scored using a semi-quantitative HSCORE, with a cut off greater than 75 considered positive., Results: Of the 60 eligible patients, 45 had evaluable tissues for all receptors. For ER, 40% of the cases were positive; for PR, 45% were positive. The sub-cellular distribution of PRA was exclusively nuclear, and 16% of the tumors demonstrated positive staining. PRB was nuclear and cytoplasmic, with 22% of the tumors staining for nuclear PRB and 36% of the tumors staining for cytoplasmic PRB. ER and PR from frozen tissues and PRA and cytoplasmic PRB from fixed tissues significantly decreased with increasing tumor grade. The co-expression of ER-alpha with PR from the frozen tissues (r=0.68, p<0.001) and PRA (r=0.58, p<0.001) from the fixed tissues was statistically significant. The ER HSCORE was related to both response and overall survival; there was no statistically significant correlation of PR with clinical response in this small number of patients., Conclusion: ER-alpha measured in metastatic endometrial carcinoma tissue prior to hormonal therapy was statistically significantly related to clinical response to daily tamoxifen and intermittent medroxyprogesterone acetate.

Background: Although the majority of endometrial cancer (EC) patients can be cured by surgery, unexpected recurrent disease may also occur in early stage patients. In the present study, whether or not the analysis of multiple biopathological parameters might lead to more accurate predictions of the clinical outcome of EC patients with long-term follow-up (FU) was investigated., Patients and Methods: Estrogen and progesterone receptor (ER and PgR) positivity and HER2 overexpression by immunohistochemistry were evaluated. The peritoneal washings (PWs) were analyzed by cytology and immunocytochemistry employing AR-3 and B72.3 monoclonal antibodies., Results: The patients with positive PW and HER2 positive tumors showed shorter overall survival compared to those bearing HER2 negative tumors (p =0.004). HER2 overexpression also influenced the patient outcome in the group with tumors lacking PgR (p = 0.004). At multivariate analysis PgR and HER2 overexpression emerged as independent prognostic factors., Conclusion: The combined analysis of these biopathological markers could provide useful information for the selection of patients to be enrolled in innovative therapeutic strategies.

Estrogen receptor beta (ERbeta) has five carboxyl-terminal (C-terminal) isoforms derived from alternative splicing. ERbeta1 is the wild-type receptor whereas ERbeta2/betacx lacks the activation function (AF)-2 core essential for ligand-dependent transcriptional activation and so behaves as a dominant-negative receptor affecting the function of ERalpha. The objective of this study was to analyze the expression of ERbeta1 and ERbeta2/betacx isoforms in nonneoplastic endometrium and endometrioid carcinoma. The study was conducted on samples of 22 proliferative endometrium, 15 secretory endometrium, 20 simple hyperplasia (without atypia), and 26 endometrioid carcinomas. The transcript and protein levels were determined by semiquantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry, respectively. For the detection of ERbeta2/betacx protein, a polyclonal antibody was raised to its unique C-terminus, characterized, and used in immunohistochemistry. The two ERbeta isoforms are expressed in the proliferative and secretory phase endometrium with no significant change in their relative levels. The levels of the ERbeta1 isoform were lower as compared to the levels of ERbeta2 in all the groups studied. Expression of ERbeta2/betacx was decreased in endometrioid carcinoma as compared to proliferative endometrium (P < 0.01). A significant decrease of the ERbeta2/ERbetacx transcript was observed with higher grade tumors (P = 0.041). Progesterone receptor (PR) expression was not influenced by either of the ERbeta isoforms which was observed by logistic regression analysis in all the groups. The coexpression of ERbeta2/betacx with ERalpha did not affect PR levels (logistic regression analysis). Thus, we conclude in the human endometrium, there is significant ERbeta2/betacx isoform expression and alterations in its levels could be involved in endometrial cancer progression.

Background and Aim: Endometrial carcinoma is the most common female pelvic genital malignancy. The strong association between the development of endometrial cancer and influence of steroid hormones (especially estrogen) was demonstrated in many studies. Mucinous carcinoma is an uncommon type of endometrial carcinoma. Most cancers are low grade and have a relatively good prognosis. The expression of one type of estrogen (ER) and progesterone receptor (PR) has been well documented. Recently, two new types of receptors (ER-beta and PR-B) were demonstrated. The aim of this study was to determine the expression of all four steroid receptors (ER-alpha, ER-beta, PR-A and PR-B) in human mucinous carcinoma of the endometrium., Patients and Methods: An immunohistochemical hormone receptor assay using specific monoclonal antibodies against estrogen receptors (ER-alpha, ER-beta) and progesterone receptors (PR-A and PR-B ) was used to study formalin-fixed and paraffin-embedded slides of 12 patients, diagnosed with primary endometrial mucinous carcinoma of different histological grades (G1 n = 9; G2 n = 3; G3 = 0)., Results: Three types of steroid receptors (ER-alpha, PR-A and PR-B) were frequently expressed in mucinous adenocarcinoma. ER-beta was weakly expressed in only one analyzed case. The immunohistochemical expression of PR-B demonstrated a statistically significant decrease in G1 neoplasms in comparison to G2 (p < or = 0.001)., Conclusion: We demonstrated the expression of four different steroid receptors in mucinous endometrial carcinoma. No significant differences between different histological grades of tumor with respect to ER-alpha and ER-beta expression were observed. Interestingly, a statistically significant increase in expression of PR-B in G2 neoplasms compared to G1 was demonstrated. The higher expression of PR-B in G2 tumors suggests a substantial function of progesterone, and thus progesterone receptor, in the malignant transformation of mucinous endometrial cancer. Therefore, PR-B expression might be utilized as a tumor marker to distinguish between G1 and G2 mucinous tumors. However, additional studies are necessary to evaluate whether these parameters could be used as tumor markers for endometrial cancer.

Objective: Progestin is an effective endocrine treatment for patients with atypical hyperplasia or with endometrial carcinoma that is estrogen receptor (ER) positive and progesterone receptor (PR) positive. However, long-term progestin treatment may lead to resistance. We have studied the progestin resistance phenotype that frequently develops in endometrial carcinoma., Methods: Ishikawa endometrial carcinoma cells were cultured for a long period (10 months) in the presence of the synthetic progestin medroxyprogesterone acetate (MPA), thereby generating a subline refractory to the growth-suppressive effects of MPA., Results: The MPA-resistant subline showed growth stimulation rather than inhibition after MPA treatment. Immunocytochemical analysis showed reduced ER alpha and PR-B expression and increased ER beta expression in this subline compared with parental Ishikawa cells. Progestin-resistant Ishikawa cells also showed increased expression of transforming growth factor alpha (TGFalpha), the epidermal growth factor receptor (EGFR), and EGFR tyrosine kinase (EGFR-TK); MPA treatment further stimulated the expression of TGFalpha in these cells. Additionally, progestin-resistant Ishikawa cells were highly sensitive to growth stimulation by TGFalpha and to growth inhibition by the EGFR-TK-specific inhibitor AG1478, and they showed increased dependence on TGFalpha-EGFR signaling., Conclusions: Our results suggest that prolonged treatment of endometrial carcinoma cells with MPA induces resistance to the growth-suppressive effects of MPA and enhances cancer cell proliferation. The downregulation of ER alpha and PR-B, the upregulation of ER beta, and highly activated TGF-EGFR signaling are thus likely to contribute to progestin resistance in endometrial carcinoma. Therefore, an EGFR-TK-specific inhibitor might be useful in the treatment of progestin-resistant endometrial carcinoma.

Unlabelled: Abnormalities in the TP53 gene are the most frequent genetic alterations in human cancers. The role and mechanism of TP53 mutations have been well studied in many types of human cancer. Similarly, the presence of microsatellite instability (MSI) in the DNA mismatch repair system (hMSH2) may provide evidence of faulty DNA mismatch repair. One of the most important locations of MSI is the BAT26 gene. In addition, deranged serum cytokines, especially elevated levels of the tumor necrosis factor (TNF) alpha, have been found in many gynecological conditions., Aims: The current study aimed at evaluating mutations in exons 6 and 7 of TP53 and the presence of microsatellite instability in BAT26 of the hMSH2 system in Egyptian patients with endometrial carcinoma. The study also evaluated whether there was a correlation between any of these genetic mutations/instability and the tissue expression of estrogen and progesterone receptors and the serum TNF-alpha level., Patients and Methods: The current study included 2 groups: a control group comprising 20 healthy women aged 52.21 +/- 5.80 years attending the clinic for routine checkups and 40 patients with endometrial cancer aged 55.30 +/- 6.21 years. Mutations in TP53 and BAT26 were evaluated using polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) and automated sequencing while serum TNF-alpha was measured using an ELISA technique. Estrogen and progesterone receptor expression in biopsy tissue was evaluated using immunohistochemical staining., Results: Seven of the 40 patients (17.5%) were positive for TP53 gene alterations in exon 6, while 9 patients (22.5%) were positive for TP53 alterations in exon 7. Cases positive for TP53 mutations had higher tumor stages. Ten patients (25%) showed MSI in BAT26. Nearly all patients with mutations in BAT26 had a strong family history for endometrial cancer (chi2=13.33, p<0.05). There was no positive correlation between the presence of MSI in the BAT26 gene and mutations in the TP53 gene or high serum TNF-alpha levels. Cases positive for TP53 mutations had a significantly higher level of TNF-alpha than cases negative for TP53 mutations (p<0.05). Cases showing mutations in exon 6 or 7 of TP53 showed a significantly higher intensity of immunohistochemical staining for estrogen and progesterone receptor expression in biopsy tissue than cases negative for mutations. (chi2=8.11, p<0.05)., Conclusion: Our results suggest that the development of endometrial carcinoma is probably mediated through a multi-step carcinogenesis pathway and mutation of TP53 does not necessarily result from the presence of microsatellite instability in BAT26. The high serum TNF-alpha levels detected in our patients may represent an immunological antitumor response that was particularly evident in cases positive for TP53 mutations.

Objective: To investigate the expressions of estrogen receptor (ER) and progesterone receptor (PR) subtypes in human endometrial carcinoma tissues., Methods: Thirty samples of normal endometrial tissue and 66 endometrial carcinomas were investigated using western blot assays and reverse-transcription polymerase chain reaction., Results: (1) Both ERalpha and ERbeta mRNA were significantly increased in endometrial carcinoma compared with that in normal endometrium tissues. The relative value of ERalpha and ERbeta mRNA in endometrial carcinoma was 8.00 +/- 7.77, 3.84 +/- 2.57 and that in normal endometrium tissues was 4.15 +/- 3.55, 0.41 +/- 0.29, respectively. (2) Decreased levels of PR, PRA, PRB protein and PR mRNA were associated with endometrium carcinogenesis (P < 0.05), while PRB mRNA expression and the ratio of PRA/PRB was not. (3) Expression of PRB mRNA was associated with ERbeta mRNA (r = 0.43, P < 0.01)., Conclusion: Overexpression of ER subtypes and down-regulation of PR may be involved in the pathogenesis of endometrial carcinoma, and expression of PRB mRNA is associated with ERbeta but not ERalpha mRNA.

Objective: To evaluate the safety of fertility-sparing hormonal therapy for endometrial cancer in young patients., Methods: Six patients diagnosed with endometrial adenocarcinoma grade I and had undergone progestin treatment were reviewed. Four patients failed progestin treatment and were then found at surgery to have both endometrial and ovarian cancers. A clonality assay using the human androgen receptor gene as the X-linked polymorphic marker and immunohistochemistry for steroid hormone receptor expression were used to delineate the relation between the endometrial and ovarian lesions and to explore possible causes of treatment failure., Results: The patients were followed for a mean of 48.8 months. Four of the six responded to the treatment at a mean of 3.5 months. Two of these patients had a recurrence within a mean of 4.5 months after their initial response. Two patients did not respond to progestin treatment. At surgery in those 4, both endometrial and ovarian tumors were found. All 6 are still alive, and 2 successfully delivered healthy infants. The clonality assay revealed an independent cell origin for the endometrial and ovarian lesions in 2 of the 4 women who failed progestin treatment. Progesterone receptors were absent in both endometrial and ovarian tumors in 2 of these 4 patients., Conclusion: The absence of progesterone receptors may relate to the failure of progestin treatment. The use of progestin treatment for well-differentiated early endometrial carcinoma should be cautious and requires very careful clinical evaluation before and after treatment.

Objectives: To determine if epigenetic interference can restore progesterone receptor-B (PR-B) expression in PR-B negative endometrial adenocarcinoma cell lines, and to characterize the kinetics of PR-B induction mediated by DNA methyltransferase and histone deacetylase inhibitors., Methods: The PR-B negative endometrioid cancer cell lines KLE and HEC-1B were used as study models. PR-B mRNA and protein expression levels were measured using real-time PCR and Western blot analysis, respectively. DNA methylation levels of the PR-B promoter were determined by methylation-specific PCR. Dose-response correlations and the duration of response to aza-deoxycytidine (ADC) and trichostatin A (TSA) were characterized. Cell responses to prolonged and repeated drug treatment were also examined., Results: Relatively low concentrations of ADC and TSA over a 24-h period induced PR-B expression. Furthermore, ADC and TSA acted synergistically to reactivate PR-B expression. Depending on the cell line used, PR-B mRNA was induced 10-110 fold. This elevated PR-B expression continued for 48 h after drug withdrawal. Sustained upregulation of PR-B mRNA and protein was observed during prolonged and repeated drug treatment., Conclusion: The epigenetically silenced PR-B gene remains sensitive to changes in DNA demethylation and histone acetylation in uterine adenocarcinoma cell lines. Treatment with ADC and/or TSA results in a robust and sustainable PR-B upregulation. These small molecule epigenetic modifying agents may be used to sensitize poorly differentiated, PR-B negative endometrial cancers to progestational therapy.

Objective: We aimed at investigating by immunohistochemistry the relationship between cyclooxygenase-2 (COX-2) and estrogen (ER), and progesterone (PR) receptors in a single institution series of 90 primary untreated endometrial cancer patients. The simultaneous assessment of p53 protein, ki67, and neu protein has been carried out., Methods: Immunohistochemistry was performed on paraffin-embedded sections by using rabbit polyclonal antiserum against human COX-2, anti-ER (clone 1D5), and anti-PR (clone 1A6) monoclonal antibodies, anti ki67 (clone MIB-1) and p53 (clone DO-7), and polyclonal antibody anti human c-erbB2/neu., Results: There was no difference in the distribution of COX-2, p53, and neu positive cases according to ER or PR positivity, while the percentage of ki67 positive endometrial tumors was significantly higher in ER negative versus ER positive tumors (54.5% versus 31.6%, P value = 0.044). ER and PR positive tumors showed a statistically significant association with clinicopathological parameters of better clinical outcome. There was no clear association between COX-2 positivity and any of the clinicopathological features. The percentage of ki67, p53, and neu positive tumors was found to be strictly related to more aggressive features. Only advanced stage of disease was found to be a predictor of poor prognosis (P value = 0.034). None of the biological parameters examined was shown to be associated with patient outcome., Conclusions: We showed that COX-2 expression is not correlated with ER, PR, p53, and neu, thus suggesting that COX-2-mediated activities may follow independent pathways. Our findings provide the rationale to design trials based on the combination of antihormones with inhibitors of COX-2 and neu in recurrent/metastatic endometrial cancer.

Objectives: To determine whether cyclooxygenase-2 (COX-2) expression is seen in endometrial cancer, endometrial hyperplasia, and normal endometria and whether it correlates with expression of estrogen and progesterone receptors., Methods: The study was a retrospective, IRB-approved analysis of biopsy samples from 14 patients with endometrial adenocarcinoma, 19 with endometrial hyperplasias, and 10 with normal endometrium. Excluded were samples from women with a history of pelvic radiation, NSAID use, or treatment with hormones during previous year. Immunohistochemical analyses were performed on formalin-fixed, paraffin-embedded tissues. Expression of COX-2, estrogen and progesterone receptors were scored according to the proportion of positive-staining cells: 1(+), <10%; 2(+), 10-50%; and 3(+), >50%. A score > or =2(+) was considered positive. Fisher's exact test and analysis of variance were used to compare proportions and continuous variables, respectively., Results: Overexpression of COX-2 was seen in 4 (29%) of the endometrial cancers, 6 (32%) of the endometrial hyperplasia, and 4 (20%) of the normal endometria. These differences were not statistically significant (P = 0.90). No COX-2 expression was found in stromal tissue. Of 14 endometrial cancers, 7 (50%) expressed any COX-2, with 4 (29%) having an expression score of > or =2(+). Of 19 endometrial hyperplasias, 11 (58%) expressed any COX-2; with 6 (32%) having a score of > or =2(+). All 10 normal endometria showed only 1(+) expression. No significant differences were detected in COX-2 expression by grade or stage of cancer. Although 100% and 95% of both hyperplasia and normal endometrium samples expressed in estrogen and progesterone receptors, respectively, only 71% and 79% of endometrial cancers expressed estrogen and progesterone receptors (P = 0.01). A nonparametric trend was performed to detect a relationship, between COX-2 and estrogen receptor or progesterone receptor expression; no significant trend was found., Conclusions: In this study, the immunohistochemical analysis showed a trend toward increased COX-2 expression in endometrial cancer and hyperplasia compared to normal endometria. A larger sample size is needed to confirm these results. The increased COX-2 expression in hyperplasia may signify an early step in carcinogenesis. These findings may represent an important treatment opportunity for synergism in the hormonal therapy of endometrial cancer.

Objective: Loss of expression of progesterone receptors (PR) in endometrial cancer is related to a more invasive and metastatic phenotype. In this study we aim to investigate whether selective loss of PRA or PRB affects the invasive capacity of endometrial cancer cells., Methods: cDNA microarrays were performed to compare gene expression profiles of a set of endometrial cancer sub-cell lines expressing PRA and/or PRB. In vitro invasion assays were performed to assess whether differences in gene expression between the lines were reflected by their invasive behavior., Results: It was observed that cell lines that express only PRA express higher levels of cadherins, and show a lower level of invasion compared to cell lines that express PRB. When cadherin function was inhibited in exclusively PRA-expressing cell lines, an increase of in vitro invasion was observed. In support of these findings, it was observed that in higher grade and more invasive endometrial cancer, expression of E-cadherin decreased., Conclusions: These results indicate that relative loss of PRA during progression of endometrial cancer can have a negative impact on cadherin expression, which may lead to development of a more metastatic phenotype.

Objective: Progesterone plays an important role in controlling proliferation and differentiation of the human endometrium. Because there are two progesterone receptor isoforms (PRA and PRB), it was important to generate tools to be able to study the role of these two progesterone receptors separately., Methods: Using stable transfection techniques, both human progesterone receptor isoforms (hPRA and hPRB) were reintroduced into a hPR-negative subclone of the well-differentiated endometrial cancer cell line Ishikawa. Several Ishikawa subcell lines were constructed, each expressing different levels of hPRA, hPRB, or hPRA and hPRB, respectively., Results: These Ishikawa subcell lines showed a marked progesterone-induced growth inhibition with induction of apoptosis after long-term culture in the presence of hormone. Upon measuring gene regulation, a clear difference in regulation of expression of the selected genes by progesterone treatment was observed between the PRA-, PRB-, or PRA/B-expressing cell lines. Integrin beta4 (ITGB4) was only regulated in PRA-expressing cells; amphiregulin was highly regulated in PRB-expressing cells; insulin-like growth factor binding protein 3 (IGFBP3) was only regulated in PRB- and PRA/B-expressing cells; and metallothionein 1L (MT1L) was highly regulated in PRA/B-expressing cells. Interestingly, based on literature data, these genes can be implicated in induction of apoptosis, but are modulated here in such a way that suggests induction of resistance against apoptosis., Conclusion: Reintroduction of PRs into Ishikawa cells rescued progesterone responsiveness in these cells. Furthermore, using these human endometrial cancer subcell lines, clear and distinct functional differences between the PR isoforms were observed.

In the present study, we analyzed the immunohistochemical findings for the RA receptor (RAR), retinoic X receptor (RXR) and hypermethylation of promoter-region CpG island methylation of RAR-beta2. Immunohistochemistry indicated that though RXR-alpha and -gamma were present in endometrial hyperplasia and cancer, other retinoid receptors were only weakly detected. The hypermethylation of RAR-beta2 was found in 75.0% of endometrial hyperplasia samples and 92.2% of carcinomas. No normal endometria had methylation. This evidence may point to one of the reasons why endometrial hyperplasia acquires high proliferative capacity without differentiation, and the hypermethylation of RAR-beta2 may occur in the early stage of endometrial carcinogenesis.

Objective: These studies were undertaken to characterize the subcellular localization of the two major isoforms of progesterone receptors (PR), PRA and PRB, in endometrial cancer., Methods: Immunohistochemistry, immunoprecipitation, and confocal microscopy were performed using Hec50co and KLE endometrial cancer cell models expressing PRA or PRB as a consequence of transduction. The location of PRB compared to PRA was determined, and antibodies were tested for specificity with respect to PR isoform recognition. Immunohistochemical analyses of PR expression and subcellular compartmentalization were also performed on 20 formalin-fixed endometrial cancer tumors., Results: Morphological and biochemical evaluations demonstrated that PRA is localized to the nucleus, even in the absence of progesterone. In contrast, a large proportion of PRB is cytoplasmic in the absence of ligand, but is rapidly translocated to the nucleus in the presence of progesterone. The differential distribution of PRA and PRB proved to be a hallmark of malignant and nonmalignant epithelia in 20 samples of archival endometrial tissue from women with the pre-operative diagnosis of endometrial cancer. All endometrial cancer specimens demonstrated cytoplasmic PRB in 50% or more of the cells, and five of the seven tumors that were moderately to poorly differentiated demonstrated no PRB staining in the nuclei. Nuclear PRB was significantly associated with increasing tumor differentiation (P = 0.031)., Conclusion: In the absence of ligand, PRA is nuclear and PRB is largely cytoplasmic. This suggests that PRA may exert ligand-independent nuclear effects, while PRB may have nongenomic cytoplasmic actions in endometrial cancer cells.

Objective: To determine PRA and PRB expressions at mRNA and protein levels in endometrial carcinomas, and to correlate the PRA and PRB expressions in tumours with clinical features including grading, surgical stage, degree of myometrial invasion, and lymph node status., Methods: Sixty-six endometrial carcinomas were investigated with Western blot assays and reverse-transcription polymerase chain reaction. The corresponding clinical information was collected. Spearman's rank correlation was used to test for association between expressions of total PR and PR isoforms in endometrial carcinomas and clinical features., Results: At protein level, expressions of PRA and PRB were significantly inversely related to grade(r=-0.343, r=- 0.310, P<0.05), and only PRA was inversely related to surgical stage(r=-0.294, P<0.05). Lymph node metastasis was associated with reduced PRA: PRB ratio(r=-0.376, P=0.044), PRA:PRB ratio was lower in the cases with lymph node metastasis compared with that without lymph node metastasis(P=0.047). No correlation was found between protein levels of isoforms of progesterone receptor and degree of myometrial invasion. PR and PRB mRNA expressions were not related to any of the clinical features discussed above., Conclusion: PRA and PRB protein levels were correlated with grade, but were not correlated with degree of myometrial invasion. Only PRA protein level was related to surgical stage. Decreased ratio of PRA: PRB was related to lymph node metastasis. PR and PRB mRNA levels had no relationship with any of the clinical features discussed above.

Endometrial carcinomas are the most common malignancy of the female genital tract and the third most common cancer in women. Progesterone and oestrogen receptors (PRs, ERs) are the most widely documented prognostic and predictive factors in endometrioid adenocarcinoma. Besides the hormonal pathway involved in the progression of preneoplastic and neoplastic lesions, alterations of the DNA methylation status have been shown to be an early signal of tumorigenesis. In this study, we show that in normal endometrium, during the proliferative phase, DNA methylation and PR expression are high, with a significant decline towards the end of the secretory phase and a gradual increase in non-atypical and atypical endometrial hyperplasia; they reach their highest level in grade I, then decrease significantly in grade-II and grade-III endometrioid adenocarcinomas. During each stage, a significant positive correlation is observed between DNA methylation and PR (P<0.0001). The strong parallelism between DNA methylation and PR expression precludes establishing a precise determination regarding the timing of these events, clearly involved in the genesis of endometrioid adenocarcinoma.

Objective: Nuclear factor kappa B (NFkappaB) is a strong anti-apoptotic factor, which is constitutively active in human endometrial cancer cells. Progesterone is the principal growth inhibitory hormone in the endometrial epithelium and promotes apoptosis. To identify the pathways through which progesterone controls NFkappaB function, we explored its genomic and non-genomic effects in endometrial cancer cells., Methods: PR-negative Hec50co endometrial cancer cells were engineered to express high levels of the A or B isoform of PR (PRA or PRB) by adenoviral infection. Cells were treated with progesterone or vehicle alone, and RNA was isolated. Affymetrix microarrays were performed and transcriptional control of the genes of highest interest was confirmed by semi-quantitative RT-PCR. To assess the non-genomic effects of PR on inflammation associated with NF-kappaB, electromobility shift assays (EMSAs) were performed., Results: Expression analysis demonstrated a significant effect of progesterone after 12- and 24-h treatment on several genes; in particular, A20 and ABIN-2 were induced through PRB. These factors bind in a complex and inhibit NFkappaB transcriptional activity. In addition, EMSAs revealed the complete inhibition of NFkappaB dimer binding to DNA by both PRA and PRB., Conclusions: Progesterone is the principal differentiating hormone in the endometrium. We have now identified several down-stream pathways of action, one of which is the control of genes involved in NFkappaB activity. The tumorigenic inflammatory and anti-apoptotic effects of NFkappaB are inhibited by progesterone/PRB through the transcriptional control of binding proteins A20 and ABIN-2. This pathway offers interesting targets for future therapeutic development.

Objectives: Progesterone receptor (PR) has been recognized as an important factor that correlates with success of endocrine treatment and patients' prognoses in endometrial cancers (EC). This study was designed to determine the clinical implications of expression of PR form A (PR-A) and B (PR-B) in EC., Methods: We have quantified the mRNA levels of total PR (PR-AB) and PR-B by real-time RT-PCR in 120 EC and 40 normal endometria (NE). We then analyzed the correlation between expression levels of each receptor and clinical characteristics including clinical stages, histological grades, depth of myometrial invasion and patients' prognoses., Results: Although the expression level of PR-B is correlated with that of PR-A, the expression pattern of PR-B classified according to clinical characteristics was different from that of PR-A. We found that high PR-B mRNA levels but not PR-A mRNA levels correlated significantly with survival, independent of clinical characteristics including clinical stages, histological grades and depth of myometrial invasion. Percentage of PR-B to total PR (B/AB) in EC is widely distributed and the ratios of middle B/AB group (0.15 < B/AB < 0.35) are 75% in NE, 47% in surviving cases of EC and 18% in dead cases of EC at 5 years, respectively., Conclusions: These results taken together indicate that PR-B may have a different function from PR-A and the intact coordinate expression of PR isoforms was disrupted in EC, especially in poor prognostic case. The measurement of PR isoforms has prognostic value in EC.

The progesterone receptor (PR) exists as two isoforms, PRA and PRB. In vitro studies have shown that these proteins are functionally distinct, suggesting that their relative expression can influence progesterone response. Low-grade endometrial stromal sarcoma (LGESS) is an uncommon tumor that usually expresses PR. In normal endometrial stroma, both PR isoforms are present with PRA predominant throughout the menstrual cycle. The relative expression of PRA and PRB in LGESS has not been previously reported. All nine cases of primary LGESS (seven uterine, two extrauterine) expressed PRB. Eight tumors also contained PRA and it was the predominant isoform in seven cases. These tumors had similar histopathologic appearances, whereas a case with approximately equal PR isoform expression showed features of sex cord or smooth muscle differentiation. An extrauterine tumor expressing only PRB had myxoid stroma. Recurrent tumor in two cases, which expressed predominantly PRA in the primary, contained reduced levels of PR consisting predominantly or entirely of PRB after prolonged interval progestin therapy. Most primary LGESSs showed PR isoform expression similar to normal endometrial stroma, consistent with the highly differentiated phenotype of this tumor. Variant differentiation or disease recurrence was accompanied by an altered PR isoform profile that could impact on hormone response.

Uteroglobin, first reported in 1968 as a steroid secreted in rabbit uterine fluid during early pregnancy, is a progesterone-regulated and progesterone-binding protein. There is evidence that indicates that uteroglobin is inversely correlated to neoplastic growth but its role to endometrial carcinogenesis is not known. Therefore we analyzed the expression of uteroglobin in 13 normal endometrium, 19 hyperplasia and 21 endometrial carcinoma samples and the relation to estrogen receptor-alpha (ER-alpha) and progesterone receptor (PR) by immunohistochemistry and Western blotting. We also analyzed the expression of uteroglobin in 15 menopausal women who received hormone replacement therapy (HRT). The expression of uteroglobin was higher during the secretory phase than in the proliferative phase; however, it was detected in endometrial hyperplasia as weakly as in the proliferative phase and decreased according to the loss of differentiation in endometrial carcinoma. The results were basically in accord with those for PR; however, the expression of uteroglobin was weak, though PR was most detected in endometrial hyperplasia. In menopausal endometrium, the group treated with estrogen plus progesterone exhibited higher expression of uteroglobin than the group treated only with estrogen. The evidence suggests that uteroglobin expression is regulated by progesterone in the normal endometrium but that the regulation by PR is lost in endometrial hyperplasia and carcinoma according to acquirement of tumorigenesis and that estrogen plus progesterone therapy reduces the risk for endometrial carcinoma by restoring uteroglobin., (Copyright 2003 Wiley-Liss, Inc.)

Objective: Expression analysis of estrogen receptor-beta (ER-beta) and estrogen receptor-alpha (ER-alpha) in tamoxifen-associated malignant endometrial tumors (TAMET) has not previously been published. Antiestrogens complexed with ER-beta have been reported to result in activation of the activator protein-1 (AP-1) pathway that may result in cell proliferation and tumor growth. In this study, the pathologic features and expression of ER-alpha, ER-beta and progesterone receptor (PR) in TAMET were determined and compared to matched cases of non-tamoxifen-associated endometrial cancers., Methods: TAMET (n = 33) were evaluated for pathologic features (tumor type, grade, depth of myometrial invasion, lymphvascular space invasion and lymph node status), expression of ER-alpha, ER-beta and PR, and survival data (mean follow-up: 28.7 months). Each case was matched to two control patients with spontaneous endometrial cancers according to tumor type, grade and stage as well as patient age and weight (mean follow-up: 51.5 months). Formalin-fixed paraffin-embedded tissue sections were immunostained with anti-ER-alpha (1D5, Dako, Carpinteria, CA) and anti-PR (PgR636, Dako). Expression scores were determined as a sum of the product of staining intensity and proportion of cells staining (H-score). Deparaffinized sections of tumor were microdissected followed by RNA isolation. Quantification of ER-beta mRNA was performed by real-time quantitative RT-PCR with results expressed as a percentage of beta-actin mRNA., Results: Of the 33 cases 20 were endometrioid (8 grade 1, 10 grade 2, 2 grade 3), 9 papillary serous and 4 malignant mullerian mixed tumors. Using a multivariate conditional regression model, TAMET had lower ER-alpha expression (P = 0.018), higher PR expression (P = 0.029), and more frequent expression of ER-beta (P = 0.032) as compared to control cases. Cases with TAMET had more deaths from cancer and significantly worse survival from disease than controls (P = 0.01 by a log rank test)., Conclusion: TAMET are characterized by a lower expression of ER-alpha, higher expression of PR and more frequent expression of ER-beta as compared to spontaneous tumors. Differential expression of ER-alpha and ER-beta may alter the expression of key target genes (such as those induced by AP-1-dependent gene transcription), and contribute to the pathogenesis and clinical behavior of these tumors. Survival from disease was significantly worse for cases with TAMET as compared to controls.