Endometrial Adenocarcinoma Specimens Research Articles

Immunohistochemical Expression of Cyclin D1 and PIN1 in Endometrial Carcinoma

Background: Cyclin D1, a positive regular of the cell cycle, may lead to uncontrolled cell proliferation. Overexpression of Cyclin D1 has been associated with the diagnosis and prognosis of numerous tumors. PIN1 binds and isomerizes the phosphorylated serine/threonine–proline motif, which lead to alteration in the structure and function of proteins. The altered phosphorylated proteins by PIN1 are closely linked to development of cancer. PIN1 is strongly expressed in the majority of tumors, suggesting it promotes tumorigenesis, and negatively associated with the clinical prognosis. Objectives: To assess Cyclin D1 & PIN1 expression and correlation in endometrial adenocarcinoma. Also to assess the relationship between Cyclin D1 & PIN1 expression and clinico-pathological variables of cases of endometrial carcinoma.Materials and Methods: Study included 30 cases of endometrial adenocarcinoma specimens. Immunohistochemical staining was performed for both Cyclin D1 and PIN1. Blocks of tumor tissue and clinical data were gathered from the files of Pathology Department of Al-Zahraa University Hospital, between July 2017 and October 2019. Results: Cyclin D1 positive expression and PIN1 high expression were increased significantly with age, high clinical stage, high pathological grade and more myometrium invasion depth. Cyclin D1 expression was positively associated with PIN1 expression (P-value = 0.004).Conclusions: Cyclin D1 & PIN1 expression associated with age, stage, grade and depth of myometrial wall invasion in patients with endometrial carcinoma. The overexpression of Cyclin D1 & PIN1 seems to indicate a more malignant phenotype of endometrial carcinoma.

PME-1 Modulates Protein Phosphatase 2A Activity to Promote the Malignant Phenotype of Endometrial Cancer Cells

Protein phosphatase 2A (PP2A) negatively regulates tumorigenic signaling pathways, in part, by supporting the function of tumor suppressors like p53. The PP2A methylesterase PME-1 limits the activity of PP2A by demethylating its catalytic subunit. Here, we report the finding that PME-1 overexpression correlates with increased cell proliferation and invasive phenotypes in endometrial adenocarcinoma cells, where it helps maintain activated ERK and Akt by inhibiting PP2A. We obtained evidence that PME-1 could bind and regulate protein phosphatase 4 (PP4), a tumor-promoting protein, but not the related protein phosphatase 6 (PP6). When the PP2A, PP4, or PP6 catalytic subunits were overexpressed, inhibiting PME-1 was sufficient to limit cell proliferation. In clinical specimens of endometrial adenocarcinoma, PME-1 levels were increased and we found that PME-1 overexpression was sufficient to drive tumor growth in a xenograft model of the disease. Our findings identify PME-1 as a modifier of malignant development and suggest its candidacy as a diagnostic marker and as a therapeutic target in endometrial cancer.

TLR3 and TLR4 expression in healthy and diseased human endometrium

BackgroundToll-like receptors (TLRs) play an essential role in the innate immune system by initiating and directing immune response to pathogens. TLRs are expressed in the human endometrium and their regulation might be crucial for the pathogenesis of endometrial diseases.MethodsTLR3 and TLR4 expression was investigated during the menstrual cycle and in postmenopausal endometrium considering peritoneal endometriosis, hyperplasia, and endometrial adenocarcinoma specimens (grade 1 to 3). The expression studies applied quantitative RT-PCR and immunolabelling of both proteins.ResultsTLR3 and TLR4 proteins were mostly localised to the glandular and luminal epithelium. In addition, TLR4 was present on endometrial dendritic cells, monocytes and macrophages. TLR3 and TLR4 mRNA levels did not show significant changes during the menstrual cycle. In patients with peritoneal endometriosis, TLR3 and TLR4 mRNA expression decreased significantly in proliferative diseased endometrium compared to controls. Interestingly, ectopic endometriotic lesions showed a significant increase of TLR3 und TLR4 mRNA expression compared to corresponding eutopic tissues, indicating a local gain of TLR expression. Endometrial hyperplasia and adenocarcinoma revealed significantly reduced receptor levels when compared with postmenopausal controls. The lowest TLR expression levels were determined in poor differentiated carcinoma (grade 3).ConclusionOur data suggest an involvement of TLR3 and TLR4 in endometrial diseases as demonstrated by altered expression levels in endometriosis and endometrial cancer.

Expression of Bcl-2 and Ki-67 in Tamoxifen-Associated Endometrial Polyps: Comparison with Postmenopausal Polyps

Background: The aim of this study was to evaluate theexpression of Bcl-2 and Ki-67 in tamoxifen (TAM)-associatedendometrial polyps and postmenopausal polyps.Material and Methods: For this purpose, a retrospectiveanalysis of paraffin-embedded specimens was carriedout. Polyps of 20 postmenopausal and 14 TAM-treatedpatients, 11 simple endometrial hyperplasia, 10 atypicalcomplex endometrial hyperplasia and 8 endometrial adenocarcinomaspecimens were included in the study.Hematoxylin/eosin-stained sections were evaluated. Immunohistochemicalstaining was performed to investigatethe expression of Bcl-2 protein and the Ki-67 proliferationindex. Results: There was no statistically significantdifference between the 5 groups with regard to Bcl-2 staining (p > 0.05). However, Bcl-2 expression inTAM-associated polyps was higher (86%) than in thepostmenopausal control group (80%). Positive Ki-67 washighest in the endometrial adenocarcinoma specimens,followed by the atypical complex endometrial hyperplasiagroup (p < 0.0001). Compared to these 2 groups, Ki-67 expression was lower in TAM-associated polyps, butKi-67 indexes were significantly higher in the TAM-associatedgroup than in the control group (p < 0.0001). Conclusion:Since TAM-associated polyps tend to have higherproliferation indexes and Ki-67 ratios than controlgroups, we suggest that they are likely to have a highermalignant potential.

THE VALUE OF HPV DNA TYPING IN THE DISTINCTION BETWEEN ADENOCARCINOMAS OF ENDOCERVICAL AND ENDOMETRIAL ORIGIN IN BIOPSY MATERIAL

Introduction and aims: Distinguishing between adenocarcinomas of endocervical and endometrial origin histologically can be difficult, particularly in curetting specimens with minimal material for examination. Endocervical adenocarcinomas have been shown to contain HPV DNA of certain ‘high risk’ subtypes, whereas this has not been consistently demonstrated in endometrial adenocarcinomas. The aims of this study were to look at whether HPV DNA typing could aid in this differential diagnosis. Methods: The study investigated the frequency of HPV DNA in paraffin embedded tissue samples from endocervical and endometrial adenocarcinoma specimens using PCR amplification techniques designed to detect HPV DNA including high risk subtypes 16, 18, 31, 33, 45 and 58. Cases were selected from PathCentre and King Edward Memorial Hospital files, mainly curetting specimens with subsequent definitive hysterectomy. All cases were reviewed by a gynaecological pathologist. Control specimens included CIN III lesions, squamous cell carcinomas (SCC's) of the cervix and lung, and colonic adenocarcinomas. Measures to prevent cross contamination were implemented. Results: HPV DNA was detected in 11 of 11 (100%) CIN III lesions, 9 of 10 (90%) cervical SCC's, 0 of 100 (0%) colorectal adenocarcinomas and 1 of 10 (10%) SCC's of the lung. 26 of 34 (76.5%) endocervical adenocarcinomas contain HPV DNA with 20 (55.6%) containing high risk subtypes, compared to 2 of 29 (6.9%) endometrial carcinomas, one with high risk subtype. Conclusions: Preliminary results suggest HPV DNA typing could be a useful adjunct in distinguishing between endocervical and endometrial adenocarcinomas on curetting specimens, and possibly in the diagnosis of metastatic carcinomas of the cervix.

Modulation of Endometrial Steroid Receptors and Growth Regulatory Genes by Tamoxifen

In Brief Objective We investigated tamoxifen's effects on the expression of growth regulatory genes in the endometrium to identify the mechanism by which tamoxifen induces proliferation. Methods Using immunohistochemical techniques, we analyzed 39 endometrial specimens for expression of Ki-67, lactoferrin, transforming growth factor-α, tumor necrosis factor receptor-II, adrenomedullin, estrogen receptors, and progesterone receptors. Twenty specimens were obtained from postmenopausal breast cancer patients treated with tamoxifen (20 mg/day) for at least 6 months to include two endometrial adenocarcinoma specimens. Five secretory phase, three proliferative phase, and seven atrophic endometrial specimens were used as controls. In addition, four endometrial adenocarcinoma specimens were reviewed from patients not treated with tamoxifen. Intensity of immunostaining was quantified using digitized imaging techniques. Results Overexpression of both estrogen receptors and progesterone receptors, and an elevated proliferative index were the most consistent effects observed in benign endometrial specimens from tamoxifen-treated patients compared with atrophic controls (P < .003). This staining pattern was also evident in adenocarcinomas from patients who received tamoxifen. Benign endometrium from tamoxifen-treated patients also expressed transforming growth factor-α, tumor necrosis factor receptor-II, lactoferrin, and adrenomedullin at levels comparable with those found in proliferative endometrial specimens. Conclusion These data provide further documentation that the uterotropic effects of tamoxifen may be due, at least in part, to the induction of estrogen receptors and progesterone receptors, as well as other genes associated with the proliferative phase. Furthermore, analysis of estrogen receptors, progesterone receptors, and Ki-67 may be useful in identifying postmenopausal individuals on tamoxifen, who are at increased risk for developing endometrial cancer. The uterotropic effects of tamoxifen may be due in part to the induction of steroid receptors and growth regulatory genes.

Modulation of endometrial steroid receptors and growth regulatory genes by tamoxifen

Objective: We investigated tamoxifen’s effects on the expression of growth regulatory genes in the endometrium to identify the mechanism by which tamoxifen induces proliferation. Methods: Using immunohistochemical techniques, we analyzed 39 endometrial specimens for expression of Ki-67, lactoferrin, transforming growth factor-α, tumor necrosis factor receptor-II, adrenomedullin, estrogen receptors, and progesterone receptors. Twenty specimens were obtained from postmenopausal breast cancer patients treated with tamoxifen (20 mg/day) for at least 6 months to include two endometrial adenocarcinoma specimens. Five secretory phase, three proliferative phase, and seven atrophic endometrial specimens were used as controls. In addition, four endometrial adenocarcinoma specimens were reviewed from patients not treated with tamoxifen. Intensity of immunostaining was quantified using digitized imaging techniques. Results: Overexpression of both estrogen receptors and progesterone receptors, and an elevated proliferative index were the most consistent effects observed in benign endometrial specimens from tamoxifen-treated patients compared with atrophic controls ( P < .003). This staining pattern was also evident in adenocarcinomas from patients who received tamoxifen. Benign endometrium from tamoxifen-treated patients also expressed transforming growth factor-α, tumor necrosis factor receptor-II, lactoferrin, and adrenomedullin at levels comparable with those found in proliferative endometrial specimens. Conclusion: These data provide further documentation that the uterotropic effects of tamoxifen may be due, at least in part, to the induction of estrogen receptors and progesterone receptors, as well as other genes associated with the proliferative phase. Furthermore, analysis of estrogen receptors, progesterone receptors, and Ki-67 may be useful in identifying postmenopausal individuals on tamoxifen, who are at increased risk for developing endometrial cancer. (Obstet Gynecol 2000;95:697–703.)

Hepatocyte Growth Factor (HGF) Induces Invasion of Endometrial Carcinoma Cell Linesin Vitro

Objectives.The overall goal of this study was to investigate the role of the hepatocyte growth factor (HGF)/Met pathway in the pathophysiology of invasive endometrial carcinoma. Our objectives were (1) to examine expression of HGF and Met in surgical endometrial carcinoma specimens and endometrial carcinoma cell lines, and (2) to determine if HGF would stimulate invasion of endometrial carcinoma cell linesin vitro.Methods.Using RT-PCR and Western immunoblotting, endometrial carcinoma specimens and the endometrial carcinoma cell lines KLE, HEC-1A, HEC-1B, and RL-95 were examined for expression of HGF and Met. A Boyden chamber invasion assay using collagen type I coated 8-μm porous membranes was then used to determine if HGF would stimulate cell invasion. Last, we assessed the capacity of endometrial stromal cells, isolated from normal human endometrium, to produce HGF as determined by an enzyme-linked immunosorbent assay and to stimulate invasion of the KLE cell line.Results.All of the endometrial carcinoma tissue samples were found to express Met mRNA, and two of four samples expressed HGF mRNA. However, the endometrial carcinoma cell lines expressed only Met and not HGF mRNA. Both the endometrial carcinoma tissue specimens and the endometrial carcinoma cell lines expressed the 140-kDa Met protein. HGF induced the invasion of the KLE and HEC-1A cells through the collagen-coated membranes in a dose-dependent fashion. The optimal concentration of HGF was between 10 and 100 ng/ml. HGF (10 ng/ml) stimulated KLE invasion 1.8-fold (P< 0.05) and HEC-1A invasion 6.5-fold (P< 0.05). During exposure to endometrial stromal cell conditioned medium containing HGF as determined by ELISA, invasion of the KLE cell line was stimulated 2.5-fold (P< 0.05).Conclusion.These results demonstrate that HGF stimulates the invasion of endometrial carcinoma cellsin vitro.Since endometrial adenocarcinoma specimens express Met, these findings suggest that the HGF/Met pathway may play a role in the invasive progression of endometrial carcinoma.

Expression of HOXD10 gene in normal endometrium and endometrial adenocarcinoma.

Hox genes encode DNA transcription regulatory proteins that contain a conserved 61 amino acid protein called the homeodomain. Although best known for their role in cellular differentiation during embryonic development, aberrant expression of these genes has been associated with hematologic and solid neoplasms. The purpose of this study was to determine the relative expression of HOXD10 in human endometrial adenocarcinomas. mRNA was isolated from 7 normal endometrial specimens and 28 endometrial adenocarcinoma specimens. cDNA was synthesized using random hexamer primers. The expression of HOXD10 relative to beta-tubulin (internal control) was assessed by densitometric comparison of co-amplified Phosphorus-32 (32P) labeled gene products separated by agarose gel electrophoresis. Direct sequencing of purified HOXD10 polymerase chain reaction product was also performed. The sequence of the purified HOXD10 product corresponds to the known DNA sequence reported in the National Institute of Health Gene Bank. mRNA expression of HOXD10 relative to beta-tubulin is significantly lower in endometrial carcinomas than in normal endometrium. Furthermore, the ratio of HOXD10 to beta-tubulin expression varies inversely with the histologic grade of the tumor (P = .0009). Cancer is a multistep process involving the aberrant expression of genes that regulate cell growth and differentiation. Human HOXD10 gene expression is altered in endometrial carcinoma and varies with the histologic grade of differentiation. This observation supports the theory that homeobox genes play a role in oncogenesis.

Expression of HOXDIO Gene in Normal Endometrium and Endometrial Adenocarcinoma

Hox genes encode DNA transcription regulatory proteins that contain a conserved 61 amino acid protein called the homeodomain. Although best known for their role in cellular differentiation during embryonic development, aberrant expression of these genes has been associated with hematologic and solid neoplasms. The purpose of this study was to determine the relative expression of HOXD10 in human endometrial adenocarcinomas.mRNA was isolated from 7 normal endometrial specimens and 28 endometrial adenocarcinoma specimens. cDNA was synthesized using random hexamer primers. The expression of HOXD10 relative to beta-tubulin (internal control) was assessed by densitometric comparison of co-amplified Phosphorus-32 (32P) labeled gene products separated by agarose gel electrophoresis. Direct sequencing of purified HOXD10 polymerase chain reaction product was also performed.The sequence of the purified HOXD10 product corresponds to the known DNA sequence reported in the National Institute of Health Gene Bank. mRNA expression of HOXD10 relative to beta-tubulin is significantly lower in endometrial carcinomas than in normal endometrium. Furthermore, the ratio of HOXD10 to beta-tubulin expression varies inversely with the histologic grade of the tumor (P = .0009).Cancer is a multistep process involving the aberrant expression of genes that regulate cell growth and differentiation. Human HOXD10 gene expression is altered in endometrial carcinoma and varies with the histologic grade of differentiation. This observation supports the theory that homeobox genes play a role in oncogenesis.

Long-term outcome of children following maternal human parvovirus B19 infection

To determine whether modulation of insulin-like growth factor-1 and insulin-like growth factor-binding protein-1 expression underlies the uterotropic effects associated with tamoxifen therapy in postmenopausal breast cancer patients. Using immunohistochemical techniques, we analyzed 37 endometrial specimens from biopsies (n = 18) or hysterectomies (n = 19) for Ki-67, insulin-like growth factor-1, and insulin-like growth factor-binding protein-1 expression. Specifically, five secretory- and three proliferative-phase endometrial specimens were used as controls; 20 specimens (including two endometrial adenocarcinomas) were analyzed from postmenopausal breast cancer patients treated with tamoxifen (20 mg/day) for at least 6 months; and nine endometrial adenocarcinoma specimens from patients not treated with tamoxifen were studied. Intensity of immunostaining was quantified using digitized imaging techniques. Insulin-like growth factor-1 and insulin-like growth factor-1-binding protein-1 were found to be expressed in normal and neoplastic endometrium of all patients, regardless of tamoxifen treatment. However, insulin-like growth factor-1 expression varied cyclically in histologically normal endometrium, was reduced in undifferentiated endometrial tumors, and was upregulated in tamoxifen-treated specimens. Insulin-like growth factor-binding protein-1 immunostaining did not vary during the menstrual cycle, but it was reduced significantly in benign tamoxifen-exposed tissue and endometrial adenocarcinomas, regardless of degree of differentiation or tamoxifen exposure. No correlation was found between the expression of insulin-like growth factor-1 and insulin-like growth factor-binding protein-1 and the proliferative indices of the tissues examined. The expression of insulin-like growth factor-1 and insulin-like growth factor-binding protein-1 in the uterus supports an autocrine and/or paracrine role for these proteins in endometrial physiology. Although further studies are needed, our investigation suggests that altered expression of insulin-like growth factor-1 and insulin-like growth factor-binding protein-1 may contribute to the uterotropic effects of tamoxifen.

Vitamin D receptor in endometrial carcinoma and the differentiation-inducing effect of 1,25-dihydroxyvitamin D3 on endometrial carcinoma cell lines.

In view of the potential of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] as a cell-differentiation-inducing agent in endometrial cancer, the localization of the vitamin D receptor (VDR) was examined immunohistochemically in 21 endometrial adenocarcinoma specimens, and the effect of 1,25(OH)2D3 on cell growth, as well as the phenotypic changes for cell maturation after treatment with 1,25(OH)2D3, was investigated in 2 endometrial carcinoma cell lines (AMEC-1, RL95-2). The VDR was detected in 14 of the 21 endometrial carcinoma specimens. The growth of RL95-2 cells expressing VDR was inhibited to 44% when cultured with 50 nM 1,25(OH)2D3 for 6 days. In contrast, the growth of AMEC-1 cells not expressing VDR was completely uninhibited even when cultured with 100 nM 1,25(OH)2D3 for 6 days. The RL95-2 cells exposed to 50 nM 1,25(OH)2D3 for 6 days had an increasing expression for 52.5 kD or 45 kD cytokeratin polypeptide, and they became columnar with pronounced polarity and formed gland-like structures when cultured in collagen gel. These results suggest that endometrial adenocarcinoma is a target for 1,25(OH)2D3, which appears to function as a cell-differentiation-inducing agent for the treatment of endometrial cancer.

Endometrial hyperplasia and endometrial carcinoma induce a vasoproliferative response in the chick embryo chorioallantoic membrane

Ten specimens of endometrial adenocarcinoma, of endometrial hyperplasia and uterine prolapse (the latter used as controls), respectively, were grafted onto the chick embryo chorioallantoic membrane (CAM) to investigate their angiogenic activity. The vasoproliferative response was assessed four days after grafting on histologic sections by a planimetric point-count method. Microvessel counts in the CAM area under and around the implants were significantly higher in endometrial adenocarcinoma than in endometrial hyperplasia and in the latter over the controls. These findings show that endometrial hyperplasia and endometrial adenocarcinoma are angiogenic. These results confirm previous observation that angiogenic activity is both a marker of the passage between preneoplastic to neoplastic status and an event that influences tumour progression.

Basement membrane components in normal hyperplastic and neoplastic endometrium.

The major basement membrane (BM) components, laminin and type IV collagen, were studied by immunochemistry in normal, hyperplastic, and neoplastic endometrium. By immunoperoxidase technique, proliferative and secretive endometrium showed capillary and epithelial cell basement membranes with linear staining with antibodies to both laminin and type IV collagen. Immunostaining of laminin and type IV collagen showed that capillaries were surrounded by a continuous perivascular sheath of these matrices in specimens of adenomatous hyperplasia and in nearly all specimens of endometrial adenocarcinoma. Laminin and type IV collagen were found to accumulate around glandular epithelial cells of adenomatous hyperplastic endometrium, but in several specimens these linear surrounding formations were defective and discontinuous. In several areas of well-differentiated endometrial adenocarcinomas BM-like structures were found around glandular epithelial cells as shadows without staining for laminin and type IV collagen. These basement membrane components accumulate around stromal cells to encircle each cell with a gradual, progressive, and cyclic process depending on the phase of the menstrual cycle. Laminin and type IV collagen were clearly detected around stromal cells at days 20 to 22 of the menstrual cycle and more thickly at days 26 to 28. The accumulation of these matrices around stromal cells is a progesterone/progestin-related process. In the well-differentiated adenocarcinoma a mid-term treatment with progestin (Danatrol Maggioni-Winthrop, SPA, Milan, Italy) was found to be effective on laminin and type IV collagen accumulation around stromal cells.

Immunocytochemical assay of estrogen receptors in endometrial carcinoma with monoclonal antibodies. Comparison with biochemical assay

Specimens of endometrial adenocarcinoma, surgically obtained from 18 women, were analyzed for distribution of estrogen receptors by an immunocytochemical assay, employing monoclonal anti-estrophilin antibodies and the peroxidase-antiperoxidase technique. Results were compared with biochemical receptor analyses, and were in concordance in 83% of them. Marked tumor cell and tissue receptor heterogeneity were apparent with the immunocytochemical method, and a variety of patterns of nuclear staining in positive tissue samples were revealed. These results indicate that the immunohistologic method will provide a number of entirely new variables that may eventually be correlated with both clinical and pathologic features of this malignancy, and may prove to be of value in the prediction of clinical endocrine response.

Peroxidase activity in glands and stroma of human endometrium

Peroxidase activity was measured in specimens of human endometrium taken at different days of the menstrual cycle. High specific activities were more frequently found in secretory endometrium than in proliferative tissue, but the variance at each phase of the cycle was large and differences were statistically not significant. The results obtained do not support the hypothesis that peroxidase activity is determined by the levels of circulating estrogens, as is apparently true in rats. Extremely high levels of peroxidase activity were noticed in some, but not all, specimens of endometrial adenocarcinoma. Endometrial glands were separated from the stroma after collagenase digestion of normal and abnormal endometrium, and peroxidase activity was measured in each fraction. The enzyme was found to be preferentially localized in the stroma, in contrast to the reported findings of predominantly epithelial localization in the rat.

Metabolism and effects of progesterone in the human endometrial adenocarcinoma cell line HEC-1

Human endometrial adenocarcinoma cells (HEC-1 line) were incubated with 14c-progestarone. Four major labeled metabolites, 3β-hydroxy 5α-pregnan-20-one, 5α-pregnane-3β,20α-diol, 20α-hydroxy-4-pregnen- 3-one and 5α-pregnane-3,20-dione were separatedby thin layer chromatography, further purified by high pressure liquid chromatography and finally identified by addition of carriers and crystallization to constant specific activity. Among these metabolites, 5α-pregnane- 3β, 20α-diol seems characteristic of this cell line since its formation from labeled progesterone was not detected in normal endometrium or in 2 specimens of endometrial adenocarcinoma. The growth of HEC cells was unaffected by either progesterone or medroxyprogesterone acetate, a slowly metabolized progestin, at about 1C −6 M levels but was inhibited by about 10 −5 M concentrations of these compounds.