Normal Proliferative Endometrium Research Articles

Cyclin D1 is significantly associated with stage of tumor and predicts poor survival in endometrial carcinoma patients.

Cyclin D1 overexpression has been described to have oncogenic role and association with diagnosis, prognosis and survival in various tumors. This study will describe the immunohistochemical phenotype of cyclin D1, and investigate the correlation between these patterns of expression and clinicopathological parameters of endometrial carcinomas, to conclude the clinical relevance of cyclin D1 expression in the evolution of endometrial neoplasms. This study employed 101 endometrial tissue samples which include 71 endometrial carcinomas and thirty normal and benign endometrium cases. All these tissue samples were used in the assembly of tissue microarrays which have been utilized afterward in immunohistochemistry staining to detect cyclin D1 expression. Forty (56.3%) cases of endometrial carcinomas showed brown nuclear expression of cyclin D1 including 36 (61%) cases of endometrioid carcinomas, and 3 (33.3%) cases of serous carcinomas. Twenty three (76.6%) cases of control group demonstrated nuclear expression. High score cyclin D1 immunohistochemical staining has been significantly linked with patient age (P=0.0001). Large proportion of high score cyclin D1 immunohistochemical staining was observed in females who are <40years of age while high proportions of negative staining were observed in older age groups. Histologic type of tissue was also significantly related to cyclin D1 immunohistochemical staining (P-value=0.0001), high staining is more common in normal proliferative and secretory endometrium while serous carcinoma is more prevalent with negative staining. Stage of tumor was significantly associated with cyclin D1 immunohistochemical staining (P-value=0.029), proportion of stage III and IV are higher in negative cyclin D1 immunostaining. Significantly higher proportion of high score cyclin D1 immunostaining is observed in controls while higher proportion of negative cyclin D1 immunostaining is observed among carcinoma cases (P-value=0.0001). No significant associations between cyclin D1 immunohistochemical staining and grade, recurrence and alive status were observed. Significant different survival distributions were observed (P-value=0.011) and poor survival behavior was correlated with negative cyclin D1 immunohistochemical staining. In conclusion, greater frequency of cyclin D1 expression was revealed in normal endometrial tissues in comparison with carcinomas. The distribution pattern of cyclin D1 immunoexpression suggests poor prognoses in endometrial carcinoma patients.

New concepts for an old problem: the diagnosis of endometrial hyperplasia.

BACKGROUNDEndometrial hyperplasia (EH) is a uterine pathology representing a spectrum of morphological endometrial alterations. It is predominantly characterized by an increase in the endometrial gland-to-stroma ratio when compared to normal proliferative endometrium. The clinical significance of EH lies in the associated risk of progression to endometrioid endometrial cancer (EC) and ‘atypical’ forms of EH are regarded as premalignant lesions. Traditional histopathological classification systems for EH exhibit wide and varying degrees of diagnostic reproducibility and, as a consequence, standardized patient management can be challenging.OBJECTIVE AND RATIONALEEC is the most common gynaecological malignancy in developed countries. The incidence of EC is rising, with alarming increases described in the 40–44-year-old age group. This review appraises the current EH classification systems used to stratify women at risk of malignant progression to EC. In addition, we summarize the evidence base regarding the use of immunohistochemical biomarkers for EH and discuss an emerging role for genomic analysis.SEARCH METHODSPubMed, Medline and the Cochrane Database were searched for original peer-reviewed primary and review articles, from January 2000 to January 2016. The following search terms were used: ‘endometrial hyperplasia’, ‘endometrial intraepithelial neoplasia’, ‘atypical hyperplasia’, ‘complex atypical hyperplasia’, ‘biomarker’, ‘immunohistochemistry’, ‘progression’, ‘genomic’, ‘classification’ and ‘stratification’.OUTCOMESRecent changes to EH classification reflect our current understanding of the genesis of endometrioid ECs. The concept of endometrial intraepithelial neoplasia (EIN) as a mutationally activated, monoclonal pre-malignancy represents a fundamental shift from the previously held notion that unopposed oestrogenic stimulation causes ever-increasing hyperplastic proliferation, with accumulating cytological atypia that imperceptibly leads to the development of endometrioid EC. Our review highlights several key biomarker candidates that have been described as both diagnostic tools for EH and markers of progression to EC. We propose that, moving forwards, a ‘panel’ approach of combinations of the immunohistochemical biomarkers described in this review may be more informative since no single candidate can currently fill the entire role.WIDER IMPLICATIONSEC has historically been considered a predominantly postmenopausal disease. Owing in part to the current unprecedented rates of obesity, we are starting to see signs of a shift towards a rising incidence of EC amongst pre- and peri-menopausal woman. This creates unique challenges both diagnostically and therapeutically. Furthering our understanding of the premalignant stages of EC development will allow us to pursue earlier diagnosis and facilitate appropriate stratification of women at risk of developing EC, permitting timely and appropriate therapeutic interventions.

Comparative characteristics of the transcriptional activity of CDH1, CTNNB1, VEGFA genes and expression of proteins E-cadherin, β-catenin and VEGFA, coded by these genes in metastatic and non-metastatic endometrioid endometrial carcinoma

The violation of Wnt-signaling pathway and cyclic neoangiogenesis is early events in endometrial carcinogenesis. In this process an important role is played by epigenetic modifications and mutations of CDH1, CTNNB1, VEGFA genes, followed by expression violations of E-cadherin, β-catenin, VEGFA encoded by them. The aim . To study the expression levels of mRNA of CDH1, CTNNB1, VEGFA genes and the expression levels of E-cadherin, β-catenin, VEGFA in non-metastatic endometrioid endometrial carcinoma (EEC) and in EEC with metastases to regional lymph nodes. Materials and methods. Immunohistochemical study of invasive рТ 1-4 N 0 M x endometrioid endometrial carcinoma (EEC) (n=56; age – 42–83 years), invasive рТ 1-4 N 1-2 M x EEC (n=30; age – 48–79 years), samples of normal proliferative endometrium (PE) (n=30; age – 41–62 years) was performed. Molecular-genetic study of invasive рТ 1-4 N 0 M x EEC (n=10; age – 45–67 years), invasive рТ 1-4 N 1-2 M x EEC (n=10; age – 44–63 years), samples of PE (n=10; age – 46–59 years) was performed. Results. EEC is characterized by the lower mRNA expression level of CDH1 gene and by the higher mRNA expression levels of CTNNB1, VEGFA genes in comparison with the PE. E-cadherin expression level is 32.6 % lower in non-metastatic EEC and also is 58.10 % lower in metastatic EEC in comparison with the PE. β-catenin expression level is 32.56 % lower in non-metastatic EEC and also is 58.11 % lower in metastatic EEC in comparison with the PE. VEGFA expression level is 27.72 % higher in non-metastatic EEC and also is 17.87 % higher in metastatic EEC in comparison with the PE. There is lower β-catenin expression level in metastatic EEC in comparison with non-metastatic EEC. Non-metastatic and metastatic EEC is characterized by the direct medium connections between the mRNA expression levels of CTNNB1 and VEGFA genes (γ=0.44), between the mRNA expression level of CTNNB1 gene and VEGFA expression level in the tumor (γ=0.37). Conclusions . These changes contribute to tumor growth, invasion and metastasis.

Morphometric analysis of endometrial cells in liquid-based cervical cytology samples.

Exfoliated endometrial cells can be seen in cervical smears in association with a wide variety of conditions ranging from normal proliferative endometrium to endometrial malignancies. It is often difficult to differentiate between benign, atypical and malignant endometrial cells using cytomorphology alone. This study was conducted to evaluate if morphometric analysis of endometrial nuclei on liquid-based cervical samples could be of help in differentiating between these endometrial cells. Three groups of cervical samples with histopathological correlation were selected: Group A: showing benign endometrial cells; Group B: showing atypical endometrial cells and Group C: showing malignant endometrial cells. There were 30 cases each in Group A and B and 39 cases in Group C. Image J, NIH, USA was used for selecting the endometrial nuclei and performing the morphometric measurements. MANOVA was used for statistical analysis. The mean nuclear area and nuclear perimeter were significantly different between the three groups of endometrial cells with a P-value <0.05. However, the mean standard deviation of the nuclear area (SDNA) was not significantly different between atypical and malignant endometrial cells (P = 0.765) and the mean nuclear diameter was not statistically different between benign and atypical cells (P = 0.112). Also, the difference in the mean integrated gray density amongst all three groups was not statistically significant (P = 0.397, 0.844, 0.732). We conclude that the morphometric parameters of the nuclear area and nuclear perimeter are helpful in differentiating between benign, atypical and malignant endometrial nuclei even in liquid-based cervical cytology samples.

Comparative immunohistochemical characteristics of expression of estrogen receptors-alpha, progesterone receptors, p16, p53, Ki-67 and caspase 3 in invasive endometrial adenocarcinoma and normal proliferative endometrium

Endometrial cancer occupies the sixth place among newly diagnosed neoplasms in women. In late stage it has unpredictable prognosis. Immunohistichemical studies of the tumor are very important for prognosis of tumor course. The aim . To characterize the expression of the estrogen receptors-alpha, progesterone receptors, p53, p16, ki-67 and caspase 3 in the invasive endometrioid endometrial adenocarcinoma compared with the normal proliferative endometrium. Materials and methods. The uteruses of 56 women with the verified diagnosis of endometrial cancer (stage pT1-4MxNx) and biopsies of 30 women with normal proliferetive endometrium were analyzed. For this purpose, immunohistochemical and morphometrical studies were used. Results: It was established that level of expression of estrogen receptors-alpha was significantly lower in tumor glands and stroma compared with normal proliferative endometrium: the median was 6.0 (5.0; 7.0) points for tumor glands and 7.0 (6.0; 8.0) points for glands of normal proliferative endometrium(р<0,05); for stromal compartment medians were 5.0 (4.0; 6.0) points and 7.0 (6.0; 7.0), respectively (р<0.05). The level of expression of progesterone receptors showed the similar pattern and also was significantly lower in tumor glands and stroma compared with normal tissue. Medians were 7.0 (6.0; 8.0) points for tumor glands and 8.0 (7.0; 8.0) points for normal proliferative endometrium (р<0.05). Medians of expression of progesterone receptors in stroma were 6.0 (4.0; 6.0) points for tumor and 7.0 (7.0; 7.0) for normal endometrial tissue (р<0.05). The distinctive feature of endometrial adenocarcinoma was significantly higher ratio of expression of estrogen receptors-alpha and progesterone receptors in tumor glands and stroma (medians were 1.17 and 1.33, respectively) compared with normal proliferative endometrium (medians for glands and stroma were the same and equaled 1.00). Also it was shown that levels of expression of Ki-67 (the median was 2.0 points), p53 (the median – 11.50%), p16 (the median – 57.08 CUOD) were statistically significantly higher and caspase 3 expression level was statistically significantly lower (the median – 48.54 CUOD) in cells of adenocarcinoma of endometrium compared with normal proliferative endometrium, where the medians for these markers were as follows: the median for Ki-67 was 1.0 point, p53 – 3.00%, p16 – 33.46 CUOD and caspase 3 – 65.02 CUOD. In the stromal component of tumor medians of expression of p16 and caspase 3 were 3.22 and 43.87 CUOD. This was statistically significantly lower than in normal endometrial tissue (the median for p16 – 10.33 CUOD and for caspase 3 – 53.50 CUOD, respectively). Conclusions . Invasive endometrioid adenocarcinoma of the endometrium loses the sensitivity of tumor glandular and stromal cells to harmonizing regulatory signals of estrogen and progesterone in comparison with normal proliferative endometrium. Endometrioid adenocarcinoma has increased expression levels of p53 and p16, elevated level of proliferation and a reduced level of apoptosis of tumor cells compared to cells of normal endometrium.

Endometrial Cancer-Associated FGF18 Expression Is Reduced by Bazedoxifene in Human Endometrial Stromal Cells In Vitro and in Murine Endometrium.

Endometrial cancer develops during exposure to estrogen unopposed by progesterone. Traditional formulations for menopausal hormone therapy include a progestin in women with a uterus. However, progestin exposure increases breast cancer risk in postmenopausal women. Alternatives to progestin include bazedoxifene (BZA), a selective estrogen receptor modulator, which prevents estrogen induced endometrial hyperplasia in clinical trials. Molecular mechanisms responsible for BZA's antiproliferative effect are not fully elucidated. We profiled endometrial adenocarcinoma, hyperplasia, and normal proliferative endometrium for differential expression in genes known to be regulated by estrogens or progesterone. Fibroblast growth factor (FGF)18, a paracrine growth factor promoting epithelial proliferation, was significantly increased in adenocarcinoma. Progesterone represses FGF18 by inducing heart and neural crest derivatives expressed transcript 2 (HAND2) in stromal cells. Notably, we confirmed lower HAND2 mRNA in adenocarcinoma, along with higher FGF tyrosine kinase receptor 2 and E74-like factor 5, collectively promoting FGF18 activity. We hypothesized BZA reduces epithelial proliferation by inhibiting FGF18 synthesis in stromal cells. To determine whether BZA regulates FGF18, we treated primary stromal cells with BZA or vehicle. In vitro, BZA reduced FGF18, but did not affect, HAND2. CD1 female mice received either BZA, conjugated estrogen (CE), or combined BZA/CE for 8 weeks. CE-treated mice had nearly 3-fold higher FGF18 expression. In contrast, BZA-treated mice, alone or with CE, had similar FGF18 as controls. Unexpectedly, BZA, alone or with CE, reduced HAND2 more than 80%, differing from progesterone regulation. Reduction of FGF18 is a potential mechanism by which BZA reduces endometrial proliferation and hyperplasia induced by estrogens. However, BZA works independently of HAND2, revealing a novel mechanism for progestin-free hormone therapy in postmenopausal women.

Immunoexpression of Cyclin D1 and PTEN in Various Endometrial Pathologies.

To determine the expression of cyclin D1 and PTEN (phosphatase and tensin homolog) in endometrial hyperplasias and neoplasias. Analytical study. The study was conducted at BMSI, JPMC, Karachi, from January 2008 to December 2012. Analysis of endometrial samples, comprising of hysterectomies and curettage, was carried out. Immunohistochemical staining was done for PTEN and cyclin D1 expression. Fifty-three endometrial samples including 23 endometrial carcinomas, 6 complex hyperplasias with atypia, 14 complex hyperplasias without atypia, 6 simple hyperplasias without atypia and 4 normal proliferative endometrium were analyzed. Fifty-two percent (12 out of 23) and 48% (11 out of 23) cases of endometrial carcinomas showed complete loss of PTEN expression and cyclin D1 over expression, respectively. Five (5 out of 6) cases of complex hyperplasias with atypia and 64.28% (9 out of 14) cases of complex hyperplasia without atypia showed complete loss of or diminished expression of PTEN whereas 66.66% (4 out of 6) cases of endometrial hyperplasia with atypia and 50% (7 out of 14) cases of endometrial hyperplasia without atypia showed cyclin D1 overexpression (p &amp;lt; 0.001). Loss of PTEN, expression and cyclin D1 overexpression was seen in a significant number of well differentiated endometrial adenocarcinomas and complex hyperplasias with atypia, suggesting both as an early event in endometrial carcinogenesis.

HPV-Associated Atypical Mitotic Figures in Squamous Intraepithelial Lesions of the Lower Female Genital Tract.

Atypical mitoses with rod and dot-shaped extra pieces of chromosomes separate to the main spindle are commonly seen in HPV-associated intraepithelial lesions. To determine the reproducibility and correlation of HPV-associated atypical mitoses (HAM) with histological diagnosis and p16 status, we performed a retrospective study. A total of 100 consecutive cervical biopsies, 10 vulvar HSIL (usual VIN), 10 differentiated VIN, 10 vulvar condylomata, 5 normal proliferative endometrial, and 5 normal ovarian follicles were assessed. The first 10 mitoses were examined in the cervical biopsies, and the case recorded as positive when one HAM was identified. The first 50 mitoses in the vulvar cases and controls were examined, and the percentage of HAM was calculated. HAM were found in 62.5% of HSIL, 14.7% of LSIL, and 0% of benign cervical biopsies. When p16 was positive, 62.7% showed HAM, and when p16 was negative, 12.5% showed HAM. HAM were commonly found in vulvar HSIL, 12% of all mitoses, but extra dots of chromosome were also occasionally found in the mitoses of differentiated VIN (1%) and rarely in normal controls (0.2%). No HAM were found in condylomata. HAM was useful to confirm SIL, but the incidence was too low for absence to exclude SIL. Although HAM are more common in HSIL, they cannot be relied upon to distinguish HSIL from LSIL. The dot form of HAM is less reliable than the rod form, as extra dots of chromosomes may be occasionally seen in differentiated VIN and rarely seen in normal proliferative endometrium.

Immunohistochemical characterization of the expression of matrix metalloproteinase - 9 (MMP-9) and tissue inhibitor of matrix metalloproteinase (TIMP-1) in the invasive endometrioid endometrial carcinoma

Endometrial carcinoma is the most common gynecological malignancy in developed countries.Aim. With the aim to determine MMP-9 and TIMP-1 expression levels in the invasive endometrioid endometrial carcinoma (EEC) cells and areas of the immunopositive cells in the center of the tumor and in the area of tumor invasion into the myometrium pathohistological, immunohistochemical and morphometric study of histological specimens of operative material of 56 women with pT1-4 stage of tumor was performed.Methods and results. It was found that MMP-9 and TIMP-1 expression occurs in 100% of invasive EEC. The ММР-9 expression level in the cytoplasm of tumor cells is high – 129.11±2.38 CUOD, which is 2,2 times more than its level in normal proliferative endometrium – 59.45±9.58 CUOD (p <0.05 ). The TIMP-1 expression level is 9.58±2.68 CUOD that is 2.69 times more than the expression level of TIMP-1 in normal proliferative endometrium (3.56±0.35 CUOD, p <0.05) . MMP-9 immunopositive cells make up 50.78±0.91% of area of invasive EEC tissue section and TIMP-1 immunopositive cells make up 3.76±0.15% of area of tumor tissue section. That is statistically significantly more than in normal proliferative endometrium, which has 23.31±1.31% of ММР-9 immunopositive cells and 2.82±0.14% of TIMP-1 immunopositive cells of endometrial glands. Differences between high levels of MMP-9 in the center of EEC and in the tumor myometrial invasion zone, as well as differences between the very low expression levels of TIMP-1 in these regions of the tumor were not statistically significant. With increasing of EEC histological grade the MMP-9 and TIMP-1 expression levels significantly increase (p <0.05). There is direct weak correlation between the MMP-9 and TIMP-1 expression levels in the cells of EEC (Pearson coefficient r =+0.31). Direct strong correlation between the expression level of MMP-9 in the tumor cells and the grade of tumor (Pearson coefficient r =+0.71) is revealed.Conclusion. The results of the study showed the increasing invasive capacity of EAE with the tumor grade increasing.

Immunohistochemical characterization of the expression of cell adhesion molecules E-cadherin and β-catenin in the invasive endometrioid endometrial carcinoma

Aim. In order to determine E-cadherin and β-catenin expression levels in the invasive endometrioid endometrial carcinoma (EEC) cells and areas of the immunopositive cells in the center of the tumor and in the area of tumor invasion into the myometrium a pathohistological and immunohistochemical study of histological specimens of operative material of 20 patients with pT1-3 stage of tumor was performed. In the comparison group the proliferative-phase endometrium of 20 women was studied. E-cadherin expression by tumor cells was determined using monoclonal antibodies E-Cadherin, Clone NCH-38 and expression of β-catenin – in the use of monoclonal antibodies Beta-Catenin, Clone E247 s-Catenin-1.Methods and results. The expression levels of immunohistochemical markers in tumor cells and tumor cells immunopositive areas were determined by photo digital morphometry in digital image processing program Image J. It was found that E-cadherin expression occurs in 95% of invasive endometrioid endometrial carcinomas and β-catenin expression takes place in 100% of EEC. Membrane-cytoplasmic expression of E-cadherin was observed in 40% of tumors, and 10% of endometrioid adenocarcinomas of the uterus with squamous differentiation showed nuclear β-catenin expression. There was a moderate expression level of E-cadherin (52,23 ± 8,59 CUOD) and high expression level of β-catenin (107,72 ± 9,73 CUOD) in the cells of invasive EEC. These levels are 2 and 5 times lower than in normal proliferative endometrium: the expression level of E-cadherin is 116,51±8,5 CUOD and β-catenin expression level is 159,7815±3,57 CUOD. E-cadherin immunopositive cells make up 20,58% ± 3,3% of area of invasive EEC tissue section and β-catenin-immunopositive cells make up 42,61 ± 4,14% of area of tumor tissue section. That is statistically significantly less than in normal proliferative endometrium, which has 100% β-catenin and E-Cadherin immunopositive cells of endometrial glands. The differences between low E-cadherin expression level in the center of EEC and moderate expression level of this marker in the region of tumor myometrial invasion, as well as differences between high expression levels of β-catenin in these areas of the tumor were not statistically significant. There is a direct strong correlation between the E-cadherin and β-catenin expression levels in the cells of EEC (Pearson correlation coefficient r = +0,73). The inverse moderate correlation is revealed between the expression level of E-cadherin in tumor cells and the degree of tumor differentiation (Pearson correlation coefficient r = –0,67).

Laminin C1 expression by uterine carcinoma cells is associated with tumor progression

ObjectivesMolecular markers associated with tumor progression in uterine carcinoma are poorly defined. In this study, we determine whether upregulation of LAMC1, a gene encoding extracellular matrix protein, laminin γ1, is associated with various uterine carcinoma subtypes and stages of tumor progression. MethodsAn analysis of the immunostaining patterns of laminin γ1 in normal endometrium, atypical hyperplasia, and a total of 150 uterine carcinomas, including low-grade and high-grade endometrioid carcinomas, uterine serous and clear cell carcinoma, was performed. Clinicopathological correlation was performed to determine biological significance. The Cancer Genome Atlas (TCGA) data set was used to validate our results. ResultsAs compared to normal proliferative and secretory endometrium, for which laminin γ1 immunoreactivity was almost undetectable, increasing laminin C1 staining intensity was observed in epithelial cells from atypical hyperplasia to low-grade endometrioid to high-grade endometrioid carcinoma, respectively. Laminin γ1 expression was significantly associated with FIGO stage, myometrial invasion, cervical/adnexal involvement, angiolymphatic invasion and lymph node metastasis. Similarly, analysis of the endometrial carcinoma data set from TCGA revealed that LAMC1 transcript levels were higher in high-grade than those in low-grade endometrioid carcinoma. Silencing LAMC1 expression by siRNAs in a high-grade endometrioid carcinoma cell line did not affect its proliferative activity but significantly suppressed cell motility and invasion in vitro. ConclusionsThese data suggest that laminin γ1 may contribute to the development and progression of uterine carcinoma, likely through enhancing tumor cell motility and invasion. Laminin γ1 warrants further investigation regarding its role as a biomarker and therapeutic target in uterine carcinoma.

Enhanced caveolin-1 expression increases migration, anchorage-independent growth and invasion of endometrial adenocarcinoma cells.

BackgroundCaveolin-1 (CAV1) has been implicated both in tumor suppression and progression, whereby the specific role appears to be context dependent. Endometrial cancer is one of the most common malignancies of the female genital tract; however, little is known about the role of CAV1 in this disease.MethodsHere, we first determined by immunohistochemistry CAV1 protein levels in normal proliferative human endometrium and endometrial tumor samples. Then using two endometrial cancer cell lines (ECC: Ishikawa and Hec-1A) we evaluated mRNA and protein levels of CAV1 by real time qPCR and Western blot analysis, respectively. The role of CAV1 expression in ECC malignancy was further studied by either inducing its expression in endometrial cancer cells with the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (4β-TPA) or decreasing expression using short-hairpin RNA constructs, and then evaluating the effects of these changes on ECC proliferation, transmigration, matrigel invasion, and colony formation in soft agar.ResultsImmunohistochemical analysis of endometrial epithelia revealed that substantially higher levels of CAV1 were present in endometrial tumors than the normal proliferative epithelium. Also, in Ishikawa and Hec-1A endometrial cancer cells CAV1 expression was readily detectable. Upon treatment with 4β-TPA CAV1 levels increased and coincided with augmented cell transmigration, matrigel invasion, as well as colony formation in soft agar. Reduction of CAV1 expression using short-hairpin RNA constructs ablated these effects in both cell types whether treated or not with 4β-TPA. Alternatively, CAV1 expression appeared not to modulate significantly proliferation of these cells.ConclusionOur study shows that elevated CAV1, observed in patients with endometrial cancer, is linked to enhanced malignancy of endometrial cancer cells, as evidenced by increased migration, invasion and anchorage-independent growth.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1477-5) contains supplementary material, which is available to authorized users.

ATM may be a protective factor in endometrial carcinogenesis with the progesterone pathway.

The purpose of the study was to explore the role and mechanism of ataxia-telangiectasia mutated (ATM) protein in endometrial carcinogenesis. A reverse-phase protein array (RPPA) was used to analyze the expression of ATM signal pathway proteins in Ishikawa and progesterone-insensitive Ishikawa. ATM expression was detected in endometrium specimens by immunohistochemistry, including 8 cases with proliferative endometrium, 6 cases with secretory endometrium, 10 cases with simple hyperplasia (SH), 13 cases of complex hyperplasia (CH), 11 cases of endometrial atypical hyperplasia (EAH), and 83 cases with type I endometrial cancer. The relationship between ATM expression and other clinicopathological indicators was also examined in type I endometrial cancer patients. The mechanisms of ATM were explored in vitro with the endometrial cell lines Ishikawa and RL95-2. A cell counting kit-8 (CCK-8) test and Western blot analysis were performed to test proliferation and protein expression. Statistical analysis was performed with SPSS19.0. The significance level was set at 0.05. ATM was increased with medroxyprogesterone acetate (MPA) stimulation in Ishikawa in RPPA. ATM expression gradually decreased in endometrial hyperplasic lesions compared with the normal proliferative and secretory endometrium and was the lowest in type I endometrial cancer. ATM expression was negatively correlated with pathological grades in type I endometrial cancer. In vitro, ATM silencing retarded proliferation inhibition in Ishikawa and RL95-2 treated with MPA. ATM silencing could down-regulate the MPA-stimulated signal proteins, including Chk2, P53, and caspase-3 in vitro. MPA might exert its role through activating the ATM-associated pathway, ATM-Chk2-P53-caspase-3 (active), preserving normal endometrium and protecting it from malignancies. ATM might be a promising indicator for endometrial hyperplasia and cancer.

Abstract 4448: Overexpression of ECT2 promotes proliferation and metastasis of UPSC

Abstract Introduction: UPSC is a rare but aggressive malignancy that accounts for no more than 5 to 10% of uterine cancers, but more than 40% of associated uterine cancer deaths. The molecular events responsible for the poor clinical outcomes observed with UPSC are largely unknown. Epithelial cell transforming sequence 2 oncogene (ECT2) is a guanine nucleotide exchange factor (Rho-GEF) that catalyzes the exchange of GDP for GTP by Rho family GTPases. High levels of ECT2 expression have been reported in brain, lung and breast cancers, where they were shown promote metastasis. However, the role of ECT2 in UPSC has not been previously explored. Methods: After obtaining IRB permission, ECT2 mRNA and protein expression were measured in flash frozen specimens of normal proliferative endometrium (n=8) and UPSC (n =8) by quantitative real-time PCR (qPCR) and Western blot. Established cultures of UPSC cell lines (UPSC-ARK1, UPSC-ARK2) were transfected with either an siRNA targeting ECT2 or a non-targeting control (Dharmacon) using Lipofectamine 2000 (Invitrogen). Reduced expression of ECT2 following transfections with ECT2 siRNAs was confirmed by qPCR and Western blot. Standard MTS and Caspase 3/7 assays (Promega) were utilized to measure proliferation and apoptosis. Colony formation and Boyden chamber assays were used to measure metastatic capacity, migration and invasion in vitro. Results: Our data indicate that ECT2 is overexpressed &amp;gt;4-fold in nearly all UPSC specimens tested (n=8, p&amp;lt;0.001). We also found that ECT2 is robustly expressed in both the UPSC-ARK1 and UPSC-ARK2 cell lines. Utilizing these 2 cell lines, we found that knockdown of ECT2 significantly decreased proliferation (UPSC-ARK1: 0.62-fold versus control; n=3, p&amp;lt;0.05; UPSC-ARK2: 0.34-fold versus control, p&amp;lt;0.05) and increased rates of apoptosis (UPSC-ARK1: 1.3-fold; n=3, p&amp;lt;0.05; UPSC-ARK2: 2.2-fold, n=3, p&amp;lt;0.05) in both cell lines tested. Decreased ECT2 expression targeted by transfection with siRNAs led to decreased rates of colony formation in UPSC-ARK1 and UPSC-ARK2 cell lines. Knockdown of ECT2 also significantly reduced rates of migration and invasion observed in vitro with both cell lines. Conclusions: Overexpression of ECT2 plays a critical role in promoting the growth and metastasis of UPSC. Targeting ECT2 expression may provide an effective strategy for improving outcomes for UPSC and other human cancers where hyperactivation of metastasis-promoting Rho GTPases are observed. Citation Format: Claire M. Mach, Thomas J. Magliaro, Matthew L. Anderson. Overexpression of ECT2 promotes proliferation and metastasis of UPSC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4448. doi:10.1158/1538-7445.AM2014-4448

ERM/ETV5 and RUNX1/AML1 expression in endometrioid adenocarcinomas of endometrium and association with neoplastic progression

The majority of endometrioid endometrial carcinomas (EEC) is diagnosed at stage I. Among these, 30% present myometrial invasion (stage IB), which is associated with tumor spread and relapse after primary treatment. Although an increased expression of RUNX1/AML1 and ERM/ETV5 in EEC have been suggested to be associated with early events of myometrial infiltration, there is no data regarding its expression along the evolution of EEC and possible associations with other clinicopathological parameters. Therefore, ERM/ETV5 and RUNX1/AML1 protein and gene expression profiles were assessed in different EEC stages to evaluate their role in endometrial carcinogenesis. RUNX1/AML1 and ERM/ETV5 proteins were analyzed by immunohistochemistry in 219 formalin fixed paraffin embedded endometrioid tumors and in 12 normal atrophic and proliferative endometrium samples. RUNX1/AML1 and ERM/ETV5 genes expression were analyzed by RT-qPCR. RUNX1/AML1 and ERM/ETV5 expression were decreased with increasing EEC stage, with a positive correlation between protein and gene expression for ERM/ETV5, but not for RUNX1/AML1. Both proteins were present in the nucleus of the tumor cells, whereas RUNX1/AML1, but not ERM/ETV5, was expressed in 7 out of 12 normal endometrial samples, with its expression being restricted to the cytoplasm of the positive cells. We concluded that there is a higher expression of ERM/ETV5 in early stages of EEC, whereas there seems to be a RUNX1/AML1 translocation from cytoplasm to nucleus in EEC neoplastic transformation.

Tumour suppressor gene and protein TP53/P53 in normal endometrium and endometrial carcinoma

Endometrial carcinoma (ECa) is the most common neoplasia of the female genital system. There are two basic types of ECa, endometrioid (estrogen related, indolent) and non-endometrioid (estrogen unrelated, aggressive). The tp53 is a tumour suppressor gene (17pl3) encoding a DNA-binding phosphoprotein P53, 53kDa, a transcription factor that mediates the cell's response to various kinds of genotoxic stress by preventing cell division and/or inducing DNA excise repair or apoptosis. Tp53 alterations results in an aberrant P53 with a longer half-life that accumulates in the cell. A total of 40 archived formalin-fixed and paraffin-embedded human biopsy tissue specimens with normal proliferative endometrium, endometrioid grade G1 and G3 and serous (SC) subtype of ECa were evaluated by immunohistochemistry for P53 expression/accumulation in nuclei of endometrial-epithelial cells; and by molecular biology methods for the exons 5-8 tp53 DNA sequence alterations. The expression/accumulation of tp53/P53 was related only to aggressive (mainly SC) types of ECa. Tp53 mutations (substitutions) were detected in SC only. There is no expression/accumulation of tp53/P53 in normal endometrium. The tp53/P53 expression/accumulation and tp53 mutations presence are associated with aggressive type of ECa. Evaluation of tp53/P53 in ECa could be a relevant component useful in research and clinical practice.

MiR-196b targets c-myc and Bcl-2 expression, inhibits proliferation and induces apoptosis in endometriotic stromal cells

What is the global expression pattern of microRNAs (miRNAs) in endometriotic stromal cells and is miR-196b involved in the pathogenesis of endometriosis? Several miRNAs are aberrantly expressed in endometriotic cyst stromal cells (ECSCs), including miR-196b whose expression is repressed in endometriotic stromal cells. Although, histologically, endometriotic tissues and normal proliferative endometrium are similar, a number of distinct molecular differences have been reported to date. The anti-apoptotic and excessive proliferative properties of endometriotic cells are considered to be involved in the development and progression of endometriosis. ECSCs and normal endometrial stromal cells (NESCs) were isolated from ovarian endometriotic tissues and eutopic endometrial tissues, respectively and compared. Aberrantly expressed miRNAs in ECSCs were identified by a global miRNA microarray technique. The roles of miR-196b in ECSC proliferation, apoptosis, and c-myc and B-cell lymphoma/leukemia (Bcl)-2 mRNA expression were investigated with precursor hsa-miR-196b transfection. The methylation status of the miR-196b gene in ECSCs and the effect of a DNA demethylating agent on miR-196b expression were also examined. miRNA microarray analysis identified eight down-regulated miRNAs (including miR-196b) and four up-regulated miRNAs in ECSCs. Compulsory expression of miR-196b directed the inhibition of proliferation and the induction of apoptosis in ECSCs. miR-196b was found to suppress c-myc and Bcl-2 mRNA expression in ECSCs, and there was a significant correlation between miR-196b and HOXA10 expression in ECSCs and NESCs. The miR-196b gene was hypermethylated in ECSCs when compared with NESCs, and the treatment with a DNA demethylating agent restored the expression of miR-196b in ECSCs. miRNA expression profiles were investigated only in the stromal component of ectopic and eutopic endometrium samples. In addition to miR-196b, the roles of other miRNAs aberrantly expressed in ECSCs should be examined. The present findings suggest that aberrant miRNA expression plays an important role in the pathogenesis of endometriosis as a part of epigenetic mechanisms, that expression of miR-196b in ECSCs is repressed by DNA hypermethylation of the miR-196b gene and this repression may be involved in the development of proliferative and anti-apoptotic characteristics of endometriosis. This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 20591920 to K.N. and no. 23592407 to H.N.) and The Uehara Memorial Foundation (to K.N.).

A Prospective Comparison of the Biopsy Results from Curettage and Hysteroscopy in Postmenopausal Uterine Bleeding

To evaluate the diagnostic accuracy of biopsy by dilatation and curettage in postmenopausal women with abnormal uterine bleeding. Prospective observational study from Mar. 2006 to Oct. 2011. University hospital. 112 postmenopausal women. Biopsy by dilatation and curettage was immediately followed by hysteroscopic biopsy and the histologic results were compared. By curettage, although performed under anesthesia, 3/3 (100%) cases of endometrial hyperplasia were reported as normal proliferative endometrium. There were two endometrial cancers (1.8%) and one case was missed as normal endometrium by curettage. Among endometrial polyps, only 3/39 (7.7%) cases were diagnosed by curettage. In postmenopausal women with abnormal uterine bleeding, biopsy by curettage may be not reliable for evaluation of endometrial pathology.

Evaluation of Endometrium for Chronic Endometritis by Using Syndecan-1 in Abnormal Uterine Bleeding

Context:Chronic endometritis is a condition observed in 3-10% of women with abnormal uterine bleeding (AUB). Diagnosis depends upon the histological detection of plasma cells within the inflammatory infiltrate in the endometrium. Plasma cells on H and E may be obscured by a mononuclear infiltrate, plasmacytoid stromal cells, abundant stromal mitosis, a pronounced predecidual reaction, menstrual features or secondary changes due to exogenous progesterone treatment prior to biopsy.Aims:The objective of this study was to determine utility of syndecan-1 in diagnosis of chronic endometritis in patients with AUB, and to see if any of the secondary histologic features in endometrial biopsy, correlated with the presence of plasma cells on immunohistochemistry (IHC).Materials and Methods:Fifty endometrial biopsies with a clinical diagnosis of AUB were taken. Endometrium in proliferative phase, secretory phase, endometrial polyps, and disordered proliferative endometrium were studied for the presence of plasma cells. IHC was done using syndecan-1. The secondary histologic features of chronic endometritis like gland architectural irregularity, spindled stroma, stromal edema and hemorrhage with the presence of plasma cells was statistically analysed. Values of P < 0.05 were considered as significant.Results:Plasma cells were seen in 11 (69%) of DPE, 8 (66%) of PEB, and 1 (7%) of normal proliferative endometrium and in 2 (40%) of secretory endometrium. Presence of stromal breakdown showed a significant association with plasma cells (P = 0.02) whereas gland architecture irregularity (P = 0.28), stromal edema (P = 0.71) and spindled stromal (P = 0.72) did not show a significant association.Conclusions:Plasma cells were significantly present in AUB patients. Syndecan-1 maybe helpful in unusual cases, where chronic endometritis is suspected as the cause of clinically significant ongoing abnormal bleeding.

PAX2 Loss by Immunohistochemistry Occurs Early and Often in Endometrial Hyperplasia

Immunohistochemical markers to assist in the diagnosis and classification of hyperplastic endometrial epithelial proliferations would be of diagnostic use. To examine the possible use of PAX2 as a marker of hyperplastic endometrium, cases of normal endometrium, simple and complex hyperplasia without atypia, atypical hyperplasia, and International Federation of Gynecology and Obstetrics (FIGO) grade 1 endometrioid carcinomas were stained for PAX2. Two hundred and six endometrial samples were available for interpretation of PAX2 staining. The percentage of cases with complete PAX2 loss (0% of cells staining) increased with increasing severity of hyperplasia: 0% of normal proliferative and secretory endometrium (n=28), 17.4% of simple hyperplasia (n=23), 59.0% of complex hyperplasia (n=83), 74.1% of atypical hyperplasia (n=54), and 73.3% of FIGO grade 1 endometrioid cancers (n=15). Partial loss of PAX2 expression did occur in normal endometrium (17.9%) but in smaller proportions of tissue and was less frequent than in simple hyperplasia (47.8% with partial loss), complex hyperplasia (32.5%), atypical hyperplasia (22.2%), and FIGO grade 1 carcinomas (20.0%). Uniform PAX2 expression was rare in complex (8.4%) and atypical hyperplasia (3.7%) and carcinoma (6.7%). When evaluating loss of PAX2 in histologically normal endometrium adjacent to lesional endometrium in a given case, statistically significant differences in staining were observed for simple hyperplasia (P=0.011), complex hyperplasia (P<0.001), atypical hyperplasia (P<0.001), and FIGO grade 1 endometrioid cancer (P=0.003). In summary, PAX2 loss seems to occur early in the development of endometrial precancers and may prove useful in some settings as a diagnostic marker in determining normal endometrium from complex and atypical hyperplasia and low-grade carcinomas. However, it is not useful in distinguishing between these diagnostic categories.