Normal Endometrial Tissue Samples Research Articles

Estrogen and estrogen receptor induce matrix metalloproteinase-26 expression in endometrial carcinoma cells

The human matrix metalloproteinase (MMP)-26, also called matrilysin-2 or endometase, has been isolated as a matrilysin (MMP-7) homolog. Several reports describe that MMP-26 may be related to the development of endometrial carcinomas. Total RNAs were isolated from 51 normal endometrial tissue samples, 6 endometrial hyperplasia tissue samples and 30 endometrial carcinomas. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate MMP-26 mRNA expression levels. We examined the effect of estrogen and its receptor (ER) on MMP-26 expression in endometrial carcinoma cell lines by real-time RT-PCR, western blot analysis and luciferase assays. To examine protein-DNA binding between ER and MMP-26 promoter, we performed chromatin immunoprecipitation (ChIP) assay. Real-time RT-PCR analysis revealed that MMP-26 mRNA expression was significantly higher in the normal human endometria and hyperplasias compared with that in endometrial carcinomas. Estrogen not only transactivated the MMP-26 promoter activity but also enhanced endogenous MMP-26 expression. The MMP-26 promoter region contains a putative ER response element (ERE). Nuclear ER protein interacted with ERE on the MMP-26 promoter by ChIP assay. We found a significant difference in MMP-26 expression in normal and malignant endometrial tissue samples and that estrogen induced MMP-26 expression. Estrogen may induce endometrial hyperplasia but not endometrial carcinoma. Our results provide evidence that regulation of MMP-26 promoter activity by estrogen may represent a mechanism for endometrial carcinogenesis.

Expression of the G protein-coupled estrogen receptor (GPER) in endometriosis: a tissue microarray study

BackgroundThe G protein-coupled estrogen receptor (GPER) is thought to be involved in non-genomic estrogen responses as well as processes such as cell proliferation and migration. In this study, we analyzed GPER expression patterns from endometriosis samples and normal endometrial tissue samples and compared these expression profiles to those of the classical sex hormone receptors.MethodsA tissue microarray, which included 74 samples from different types of endometriosis (27 ovarian, 19 peritoneal and 28 deep-infiltrating) and 30 samples from normal endometrial tissue, was used to compare the expression levels of the GPER, estrogen receptor (ER)-alpha, ER-beta and progesterone receptor (PR). The immunoreactive score (IRS) was calculated separately for epithelium and stroma as the product of the staining intensity and the percentage of positive cells. The expression levels of the hormonal receptors were dichotomized into low (IRS < 6) and high (IRS > =6) expression groups.ResultsThe mean epithelial IRS (+/−standard deviation, range) of cytoplasmic GPER expression was 1.2 (+/−1.7, 0–4) in normal endometrium and 5.1 (+/−3.5, 0–12) in endometriosis (p < 0.001), of nuclear GPER 6.4 (+/−2.6, 0–12) and 6.8 (+/−2.9, 2–12; p = 0.71), of ER-alpha 10.6 (+/−2.4, 3–12) and 9.8 (+/−3.0, 2–12; p = 0.26), of ER-beta 2.4 (+/−2.2; 0–8) and 5.6 (+/−2.6; 0–10; p < 0.001), and of PR 11.5 (+/−1.7; 3–12) and 8.1 (+/−4.5; 0–12; p < 0.001), respectively. The mean stromal IRS of nuclear GPER expression was 7.7 (+/−3.0; 2–12) in endometrium and 10.8 (+/−1.7; 6–12) in endometriosis (p < 0.001), of ER-alpha 8.7 (+/−3.1; 2–12) and 10.6 (+/−2.4; 2–12; p = 0.001), of ER-beta 1.8 (+/−2.0; 0–8) and 5.4 (+/−2.5; 0–10; p < 0.001), and of PR 11.7 (+/−0.9; 8–12) and 10.9 (+/−2.0; 3–12; p = 0.044), respectively. Cytoplasmic GPER expression was not detectable in the stroma of endometrium and endometriosis. The observed frequency of high epithelial cytoplasmic GPER expression levels was 50% (n = 30/60) in the endometriosis and none (0/30) in the normal endometrium samples (p < 0.001). High epithelial cytoplasmic GPER expression levels were more frequent in endometriomas (14/20, 70%; p = 0.01), as compared to peritoneal (9/18, 50%) or deep-infiltrating endometriotic lesions (7/22, 31.8%). The frequency of high stromal nuclear GPER expression levels was 100% (n = 74/74) in endometriosis and 76.7% (n = 23/30) in normal endometrium (p < 0.001). The frequency of high epithelial nuclear GPER expression levels did not differ between endometriosis and normal endometrium.ConclusionsThe present data indicate a unique GPER expression pattern in endometriosis, especially in endometriomas as compared to the normal endometrium. The overexpression of GPER in endometriotic lesions suggests a potential role for GPER in the hormonal regulation of endometriosis, which should be taken into consideration for future hormonal treatment strategies.

P53 is correlated with low BMI negative progesterone receptor status and recurring disease in patients with endometrial cancer

ObjectiveP53 tumor suppressor gene plays a role in endometrial carcinogenesis. Former studies described correlations between p53 protein overexpression in endometrial cancer and prognostic factors, measured by immunohistochemistry. But data is still controversial. The aim of this study was to measure p53 and phospho-p53 overexpression by Western blot and evaluate correlations between overexpression and prognostic and clinical factors. Phospho-p53 seems to be the functional p53 protein and was examined for the first time in endometrial cancer. Methods40 patients with endometrial cancer were included in the study. A control group of 20 patients with normal endometrial tissue samples was used. Western blot was performed for detection of p53 and phospho-p53. Clinical and pathological parameters were obtained from medical records. Statistical analysis was performed using the log-rank test, the Mann–Whitney test for two independent groups and the Fisher’s exact test for dichotomous groupings. ResultsIn 17.5% of the patients with endometrial cancer a p53 overexpression could be evaluated. There was a correlation between a p53 overexpression and recurring disease (p: 0.014), a negative progesterone receptor status (p: 0.021) and a low BMI (p: 0.022). Only one of 40 patients had a phospho-p53 expression. ConclusionWestern blot is a valid method for the detection of p53 overexpression. As other authors described before, p53 overexpression seems to correlate with negative prognostic factors. The correlation between p53 overexpression and a low BMI may underline the relationship between p53 alterations and biological aggressive endometrial carcinomas.

Low Ascorbate Levels Are Associated with Increased Hypoxia-Inducible Factor-1 Activity and an Aggressive Tumor Phenotype in Endometrial Cancer

Activation of the transcription factor hypoxia-inducible factor (HIF)-1 allows solid tumors to thrive under conditions of metabolic stress. Because HIF-1 is switched off by hydroxylation reactions that require ascorbate, inadequate intracellular ascorbate levels could contribute to HIF-1 overactivation. In this study, we investigated whether the ascorbate content of human endometrial tumors [known to be driven by HIF-1 and vascular endothelial growth factor (VEGF)] influenced HIF-1 activity and tumor pathology. We measured protein levels of HIF-1alpha and three downstream gene products [glucose transporter 1 (GLUT-1), Bcl-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), and VEGF], as well as the ascorbate content of tumor and patient-matched normal endometrial tissue samples. HIF-1alpha and its downstream gene products were upregulated in tumor tissue, with the highest levels being present in high-grade tumors. High-grade tumors also had reduced capacity to accumulate ascorbate compared with normal tissue; however, all grades contained tumors with low ascorbate content. Tumors with the highest HIF-1alpha protein content were ascorbate deficient. Low ascorbate levels were also associated with elevated VEGF, GLUT-1, and BNIP3 protein levels and with increased tumor size, and there was a significant association between low tissue ascorbate levels and increased activation of the HIF-1 pathway (P = 0.007). In contrast, tumors with high ascorbate levels had lesser levels of HIF-1 activation. This study shows for the first time a likely in vivo relationship between ascorbate and HIF-1, with low tumor tissue ascorbate levels being associated with high HIF-1 activation and tumor growth.

Abstract 3922: Lef-1 is differentially expressed in endometrial cancer and appears to target cyclin D1 in endometrial tissue

Abstract Objectives: The molecular pathways involving endometrial carcinogenesis and gland formation are currently unclear. We have previously reported on the involvement of Lymphoid Enhancing Factor-1 (Lef-1), a transcription factor, in the development and maintenance of glands within the mouse uterus. We have also demonstrated the overexpression of Lef-1 within human cancer samples. Cyclin D1, a downstream target of Lef-1, has been shown to be over-expressed in a myriad of human cancers. We hypothesize that Lef-1 is differentially expressed in human endometrial cancers and that cyclin D1 is regulated by Lef-1 in gland formation and cancer. Methods: Using Lef-1 knock out (KO) mice and wild type (WT) controls we characterized the expression of cyclin D1 within the endometrial glands of the developing mouse endometrium. Using Western blot analysis and real time PCR, we also characterized and identified Lef-1 expression in different human endometrial cancer samples. Using both normal and cancerous human endometrial tissue samples, we immunostained for Lef-1 and cyclin D1 protein. Results: Lef-1 expression is increased in certain subtypes of human endometrial cancer. In WT mice, cyclin D1 is localized to the developing gland bud by day 7, as compared to the KO mice, which express no cyclin D1 during this day. There appears to be cyclin D1 immunostaining within the same Lef-1 expressing cells within human endometrial cancers. Conclusions: Lef-1 is overexpressed in specific subtypes of human endometrial cancer. Cyclin D1 appears to be present during endometrial gland formation within the mouse uterus and present within certain human endometrial cancer cells that express Lef-1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3922.

DcR1 expression in endometrial carcinomas

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family, which mediates apoptosis by the extrinsic pathway. Up-regulation of decoy receptors, DcR1 and DcR2, may result in diminished binding of TRAIL to their functional receptors. DcR1 expression was assessed in normal endometrial tissue (NE) and endometrial carcinoma (EC) samples by immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (PCR). IHC was performed in two tissue microarrays; one composed of 80 samples of NE and a second one constructed from paraffin-embedded blocks of 62 EC. For quantitative real-time RT-PCR analysis, RNA was obtained from 19 NE and 28 EC samples using Trizol. mRNA expression of DcR1 was assessed with Taqman-based assays in an Abi-Prism 700 SDS. Results were correlated with stage, histological type, and grade. By IHC, cytoplasmic expression of DcR1 was frequently seen in NE (79.6%) and varied according to the menstrual cycle. Positive DcR1 immunostaining was also detected in EC (98.1% of the cases) without any specific statistical association with histological type, grade, and stage. By quantitative real-time PCR, all NE had similar levels of DcR1expression (0.8-1.7 RQ), which were considered the basal levels of DcR1 expression in NE. Increased DcR1 expression (> or =5-fold higher than the basal levels) was detected in 13 of 28 EC (46.4%). High DcR1 expression levels were found in ECs of different stages: IA, four of 12 (33%); IB, two of four (50%); IC, four of six (66%); and IIA and IIB three of six (50%). Results suggest that DcR1 expression occurs in a subset of EC and may contribute to resistance to TRAIL-induced apoptosis.

Side population cells contribute to the genesis of human endometrium

To investigate whether stem (progenitor) cells are found in human endometrial side population cells. Experimental laboratory study. University-based laboratory in Japan. Normal endometrial tissue samples from 42 patients. Side population cell analysis and staining were performed by using Hoechst 33342, CD31, CD34, EMA, CD105, CD146, and BCRP1/ABCG2. Human endometrial side population cells were isolated and characterized by fluorescence-activated cell sorter analysis. Stem cell activity was evaluated by colony-forming assays and cell cycle analysis. Endometrial side population cells expressed not only the endothelial cell markers CD31 and CD34 and the epithelial cell marker EMA but also mesenchymal stem cell markers CD105 and CD146. Immunohistochemical analysis revealed that BCRP1/ABCG2, known as a marker of side population cells, was strongly expressed in the vascular endothelium and the epithelium of the basal layer of the endometrium. In cell cycle analysis, side population cells isolated directly from tissue were mainly in G0, whereas side population cells sorted after primary culture included populations in G1 and G2/M/S. These sorted side population cells showed greater colony-forming efficiency than non-side population cells and secreted PRL in an in vitro decidualization model. Human endometrial side population cells may include putative stem or progenitor cells.

Overexpression of the osteopontin correlates with the aggressiveness of endometrial cancer

16048 Background: To test the hypothesis that expression of osteopontin (OPN), an integrin-binding glycoprotein, can independently predict the potential aggressiveness of endometrial cancer. Methods: The status of OPN expression in benign and malignant endometrial cancer cell lines and tissues was analyzed by real time PCR, Western blot, and immunohistochemistry. Nonparametric Spearman's correlation coefficient method was used to assess the statistical significance of the correlation between clinicopathologic characteristics of tumor and OPN expression. Results: An increased expression of OPN was observed in the endometrial cancer compared to normal endometrial tissue samples. When the level of OPN in normal tissue was set at 1, its level in benign endometrial hyperplasia was slightly increased at 1.2, whereas the OPN level in the highly malignant endometrial carcinoma tissue was greatly increased by nearly 3–5 folds. Amongst the 160 cases examined immunohistochemically, of the 43 grade 1 endometrial carcinomas, 31 were unstained and 12 stained weakly positive (+). For the 41 grade 3 or serous type endometrial carcinomas analyzed, 25 (60%) stained strongly positive (+++), 8 (19%) stained moderately positive (++) and 4 (9%) stained weakly positive (+). These results showed that the level of OPN expressed between grade 1 and grade 3 or more was significantly different (Spearman's correlation coefficient method, p = 0.001). However, Kaplan-Meier survival analysis showed that the increased level of OPN expression was not significantly associated with reduced survival time of the patients. Conclusion: The results suggest that the increased OPN level may be involved in the malignant transformation of endometrial adenocarcinoma cells. No significant financial relationships to disclose.

Overexpression of claudin‐3 and claudin‐4 receptors in uterine serous papillary carcinoma

Uterine serous papillary carcinoma (USPC) represents a highly aggressive variant of endometrial cancer. Using gene expression profiling, we recently identified high expression of the claudin-3 and claudin-4 receptors in a limited set of USPC. These tight junction proteins represent the low- and high-affinity receptors, respectively, for the cytotoxic Clostridium perfringens enterotoxin (CPE) and are sufficient to mediate CPE binding and trigger subsequent toxin-mediated cytolysis. The potential for targeting this pathway in the treatment of USPC was explored. Claudin-3 and claudin-4 receptor expression was analyzed at the mRNA and protein levels in flash-frozen and formalin-fixed, paraffin-embedded tissue from 20 consecutive USPC patients. The potential of recombinant CPE as a novel therapy against primary, metastatic, and chemotherapy-resistant USPC cell lines was also investigated in vitro. Finally, the in vivo therapeutic effect of sublethal doses of CPE was studied in SCID mouse xenografts harboring subcutaneous and intraperitoneal USPC that expressed claudin-3 and claudin-4. In all, 100% (20 out of 20) of the primary flash-frozen USPC tested overexpressed 1 or both CPE receptors by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). Membranous immunoreactivity for claudin-4 protein expression was documented in the majority of USPC specimens tested by immunohistochemistry, whereas only a low level of membranous staining was found in normal endometrial control tissue samples. When primary and metastatic short-term USPC cell lines were incubated with different concentrations of CPE in vitro, a dose-dependent cytotoxic effect was demonstrated. In vivo, intratumoral injections of well-tolerated doses of CPE in large subcutaneous USPC xenografts led to large areas of tumor cell necrosis and tumor disappearance in all the treated animals, whereas sublethal intraperitoneal injections of CPE had a significant inhibitory effect on tumor progression, with extended survival of animals harboring chemotherapy-resistant intra-abdominal USPC carcinomatosis. Claudin-3 and claudin-4 receptors may offer promising targets for the use of CPE as a novel type-specific therapy against this highly aggressive and chemotherapy-resistant variant of endometrial cancer.

Expression of Organic Cation Transporter SLC22A16 in Human Endometria

SLC22A16 is one of newly isolated organic cation transporters, which is responsible for uptake and transport of adriamycin into cells. Adriamycin is one of the key drugs for treatment of endometrial cancer. Therefore, we examined expression of SLC22A16 in human endometrium and its disorders. Protein and mRNA expression levels of SLC22A16 were examined in 124 endometrial cancer specimens, 25 normal endometrial tissue samples (15 in proliferative phase, 10 in secretory phase), and 7 endometrial cancer cell lines using immunohistochemical analysis and reverse transcription-polymerase chain reaction. Changes in SLC22A16 mRNA expression level after progesterone exposure were also examined. Immunohistochemical analysis showed that SLC22A16 protein was highly expressed in endometrium during the normal secretory phase, but its level was significantly reduced in the proliferative phase. SLC22A16 protein was detected in 59 of 124 (48%) endometrial cancer specimens and 3 of 7 (43%) endometrial cancer cell lines. The mRNA levels measured by quantitative reverse transcription-polymerase chain reaction were comparable with levels of protein expression. Furthermore, SLC22A16 mRNA levels were increased in endometrial cancer cell lines in the presence of progesterone. In conclusion, SLC22A16 is expressed in various endometrial tissues. Its expression level is high during the secretory phase and may be regulated by progesterone. Our findings also suggest that it may be possible to use progestins to increase the response of endometrioid endometrial carcinoma with SLC22A16 expression to adriamycin-based chemotherapeutic regimens.

Aberrant expression of deoxyribonucleic acid methyltransferases DNMT1, DNMT3A, and DNMT3B in women with endometriosis

Since endometriosis is a persistent disease with substantial gene dysregulation, there must be cellular memory of some sort that constitutes a unique cell identity for endometriotic cells. Epigenetic regulation, especially through DNA methylation, is a flexible, yet stable, mechanism for maintaining such a cellular memory. The aim of this study was to determine gene expression levels of DNMT1, DNMT3A, and DNMT3B, the three genes coding for DNA methyltransferases that are responsible for methylation. Cross-sectional measurements of gene expression levels of DNMT1, DNMT3A, and DNMT3B on endometriotic tissue. Academic. Seventeen patients with laparoscopically confirmed endometriosis and 8 healthy women who underwent tubal sterilization who were free of endometriosis were recruited for the study. Epithelial cells were harvested from tissue samples by laser capture microdissection and messenger RNA abundance was measured by quantitative real-time reverse transcription-polymerase chain reaction. The expression levels of these genes in epithelial cells from 13 ectopic endometrial tissue samples, 10 eutopic endometrial tissue samples taken from women with endometriosis, and 8 normal endometrial tissue samples from women without endometriosis. The genes DNMT1, DNMT3A, and DNMT3B were over-expressed in the ectopic endometrium as compared with normal control subjects or the eutopic endometrium of women with endometriosis, and their expression levels were correlated positively with each other. The aberrant expression of these genes suggests that aberrant methylation may be rampant in endometriosis. This also provides a strong piece of evidence that endometriosis ultimately may be an epigenetic disease.

Overexpression of the osteopontin correlates with the aggressiveness of endometrial cancer

5038 Background: To test the hypothesis that expression of osteopontin (OPN), an integrin-binding glycoprotein, can independently predict the potential aggressiveness of endometrial cancer. We studied OPN expression in endometrial cell carcinomas and correlated OPN expression levels with clinicopathologic tumor features. Methods: The status of OPN expression in benign and malignant endometrial cancer cell lines and tissues was analyzed by Western blot and immunohistochemistry. Nonparametric Spearman’s correlation coefficient method was used to assess the statistical significance of the correlation between clinicopathologic characteristics of tumor and OPN expression. Results: An increased expression of OPN was observed in the endometrial cancer compared to normal endometrial tissue samples. When the level of OPN in normal tissue was set at 1, its level in benign endometrial hyperplasia was slightly increased at 1.2, whereas the OPN level in the highly malignant endometrial carcinoma tissue was greatly increased by nearly 3- 5 folds. Amongst the 70 cases examined immunocytochemically, of the 23 grade 1 endometrial carcinomas, 6 were unstained and 12 stained weakly positive (+). For the 20 grade 3 or serous type endometrial carcinomas analyzed, 8 (40%) stained strongly positive (+++), 8 (40%) stained moderately positive (++) and 1 stained weakly positive (+). These results showed that the level of OPN expressed between grade 1 and grde 3 or more was significantly different (Spearman’s correlation coefficient method, p = 0.001). Kaplan-Meier survival analysis showed that the increased level of OPN expression was significantly associated with reduced survival time of the patients. Conclusions: The results suggest that the increased OPN level may be involved in the malignant transformation of endometrial adenocarcinoma cells and OPN expression level is an important determinant for patient survival. No significant financial relationships to disclose.

Carcinoma cell-specific Mig-7: A new potential marker for circulating and migrating cancer cells

Identification of genes that are expressed in a cancer cell-specific manner can provide markers for detection, diagnosis, and disease progression. We have previously reported that receptor tyrosine kinase ligands in concert with ligation of alphavbeta5 integrin induce expression of Mig-7 restricted to carcinoma cells. Because of this highly specific expression, we hypothesized that Mig-7 could be used as a marker of occult tumor cells. The objective of this study was to begin to test this hypothesis by generating Mig-7 specific antisera and RT-PCR methods for detection of Mig-7 expression in tissues and blood from cancer patients as compared to those from normal subjects. By immunohistochemistry and by RT-PCR, we detected Mig-7 mRNA in lymph nodes from 7 out of 9 (77.8%) endometrial carcinoma xenograft mice but not from any of the 5 negative control animals. Mig-7 expression was more specific than Met expression, the RTK that binds Scatter factor and is used as a marker of poor progression, in endometrial carcinoma as compared to normal endometrial tissue samples. In 87.3% of tumors from various tissues including breast, lung, colon and ovary, we detected Mig-7 expression. Blood samples from untreated metastatic cancer patients also displayed Mig-7 mRNA in contrast to a lack of expression in chemotherapy treated or normal individuals. In conclusion, we report the first immunohistochemical and RT-PCR assays for Mig-7 and discuss its highly specific localization to cancer cells in contrast to an absence in normal cells. Our preliminary data indicate that Mig-7 may be a potential early marker of migrating and circulating carcinoma cells.

Protein expression profiling of endometrial malignancies reveals a new tumor marker: chaperonin 10.

Endometrial carcinoma is a common malignancy in women, being exceeded in incidence only by that of breast, lung, and colorectal cancers. At present, no serum tumor markers are available for the monitoring of endometrial carcinoma patients, and patients with recurrent disease are detected only following the development of symptoms or abnormalities in imaging assessments. Similarly, no screening tools are available for endometrial carcinoma. Protein profiling by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a sensitive and fast method of analysis for small proteins or peptides to yield specific biomarkers. In this study, a variety of normal and malignant endometrial tissue samples were fractionated and analyzed by SELDI-TOF MS (SELDI is a version of MALDI utilizing protein "chips"). A number of proteins displayed differential expression in malignant endometrial tissues. One of the prominent proteins fractionated by weak cation exchange chromatography and displaying enhanced expression in these malignant tissues was identified as chaperonin 10. The increased expression of chaperonin 10 in malignant endometrial tissues was further confirmed by parallel Western blot and immunohistochemistry analyses.

Matrix metalloproteinase-26 is expressed in human endometrium but not in endometrial carcinoma.

The human matrix metalloproteinase (MMP)-26, also called matrilysin-2 or endometase, has been isolated as a matrilysin (MMP-7) homolog. Matrix metalloproteinase-26 was expressed in tissue samples from the placenta and endometrial tumors and its expression may be related to the development of endometrial carcinomas. Total RNAs were isolated from 5 endometrial carcinoma cell lines, 36 normal endometrial tissue samples, 4 hyperplasia tissue samples, and from 24 endometrial carcinoma tissue samples. Reverse transcription-polymerase chain reation (RT-PCR) was performed to detect MMP-26 mRNA expression. To identify MMP-26 mRNA localization and protein expression, we performed in situ RT-PCR and immunohistochemistry, respectively. Reverse transcription-polymerase chain reaction analysis revealed that MMP-26 mRNA was expressed in 24 of 36 normal human endometrial tissue samples. However, MMP-26 mRNA expression was not detected in endometrial carcinoma cell lines nor in endometrial carcinoma tissue samples except for one case. Western blot analysis showed similar results. In situ RT-PCR analysis revealed that MMP-26 expression was localized in the epithelial glandular cells but faint expression was observed in the stromal cells. Subsequently, we separated endometrial tissues into epithelial glandular and stromal cells. Using RT-PCR, the purified epithelial glandular cells exhibited MMP-26 mRNA expression but the purified stromal cells did not. Immunohistochemical analyses revealed that MMP-26 protein expression is also limited to endometrial epithelial glandular cells but not to cancer cells. Therefore, MMP-26 expression is limited to normal epithelial glandular cells. We found a significant difference in MMP-26 expression in normal and malignant endometrial tissue samples, although its function is still unknown. These data suggest that MMP-26 may be a candidate for a new tumor marker for endometrial carcinomas.

Clonal analysis and mutations in the PTEN and the K-ras genes in endometrial hyperplasia.

The patterns of X chromosome inactivation and mutations of PTEN and K-ras were evaluated in cases of endometrial hyperplasia to determine the presence of potentially premalignant neoplastic versus polyclonal benign cell populations. Endometrial glandular epithelial cells were collected by laser capture microdissection, and genomic DNAs were extracted. Following treatment with the methylation sensitive restriction endonuclease Hha I, polymerase chain reaction amplification was performed targeting a highly polymorphic short tandem repeat of the human androgen receptor gene (HUMARA). PTEN and K-ras gene mutations were evaluated by analysis of single-strand conformation polymorphism. Two pathologists performed histologic diagnosis of the lesions independently. Monoclonal composition was demonstrated in 13 of 15 (87%) endometrial hyperplasias with atypia and 17 of 31 (55%) complex hyperplasias without atypia. Cytological atypia is significantly associated with the clonal status of the endometrial hyperplasia (13/15 vs 17/31, P = 0.049). In contrast, all 14 normal endometrial tissue samples were polyclonal. PTEN gene mutations were detected in 4 of 13 (30%) monoclonal endometrial hyperplasias with atypia and 2 of 17 (12%) monoclonal endometrial hyperplasias without atypia but were not detected in polyclonal endometrial hyperplasias, with or without atypia. K-ras gene mutations were present in 3 of 13 (23%) monoclonal endometrial hyperplasias with atypia but not in 2 cases of polyclonal endometrial hyperplasia with atypia or in 26 cases of endometrial hyperplasia without atypia. K-ras mutation is thus significantly more frequently found in endometrial hyperplasias with atypia than those without atypia (3/15 vs 0/31, P = 0.030). This study indicates that most cases of endometrial hyperplasia with atypia and a high proportion of cases of endometrial hyperplasias without atypia originate from a single progenitor cell, possibly as a result of genetic alterations, rather than as a result of benign reactive processes.

PTEN Expression in Breast and Endometrial Cancer: Correlations with Steroid Hormone Receptor Status

Objective: The PTEN (MMAC1/TEP1) tumor suppressor gene is frequently mutated and homozygously deleted in human neoplasms, but there is only sparse information about PTEN protein expression in hormone-dependent female tumors. Therefore, we investigated PTEN expression in 68 breast and 43 endometrial carcinomas. Methods: For PTEN protein detection, we used Western blot analysis followed by densitometry and compared these data with clinicopathologic parameters, the estrogen receptor (ER) and progesterone receptor (PR) status, HER2/neu and the proliferation marker Ki67. Results: We were able to show significantly decreased PTEN protein expression in endometrial carcinomas compared with normal endometrial tissue samples, especially in the endometrioid histological subtype. In contrast, PTEN downregulation was found more rarely in breast cancer. Lower PTEN expression in breast cancer correlated significantly with high ER immunoreactivity (p = 0.008) and was weakly associated with PR expression (p = 0.055) and low histological grading (p = 0.081). No correlation with any of these parameters was observed in endometrial tumors. In both tumor types, no association of PTEN expression with any other analyzed parameter was found. Conclusions: These results suggest that PTEN expression plays different roles in the pathogenesis of endometrial carcinomas and breast cancer. In mammary carcinomas, loss of PTEN expression is mainly found in more differentiated tumors and is probably not a major event in carcinogenesis.

Expression of cell-cycle regulatory proteins in endometrial carcinomas: correlations with hormone receptor status and clinicopathologic parameters.

The normal human endometrium is characterized by hormone-dependent variations in the levels of cell-cycle regulatory proteins during the menstrual cycle. As this tightly controlled system is disturbed in endometrial carcinomas, we analyzed which cell-cycle regulators are involved in endometrial carcinogenesis. We performed Western blot analysis of five cell-cycle stimulating (cyclins D1, E, B1, cdk2, cdk4) and three cell-cycle inhibiting (p16(INK4a), p21(WAF1), Rb) proteins in 41 endometrial carcinoma specimens. In addition, expression of the estrogen and progesterone receptors (ER, PR), Ki67, and, in selected cases, p16, cyclin E, and cyclin B1 was studied by immunohistochemistry. We found upregulation of all analyzed cell-cycle regulators in most tumors compared to normal endometrial tissue samples. Overexpression of cyclin E, cyclin B1, and p21 was associated with a less differentiated phenotype. In addition, high levels of cyclin E, cdk2, and cdk4 correlated with weak/absent ER expression, and p16 and p21 overexpression was significantly associated with low PR immunoreactivity. Cyclin B1 expression correlated with cyclin E, cdk2, cdk4, p21, Rb, and Ki67, and cyclin E expression with cyclin D1 and Rb. We conclude that cyclin E and cyclin B1 might be the major cell-cycle regulators involved in proliferation and reduced differentiation of endometrial carcinomas. In addition, p16, p21, and Rb appear to be uncoupled from their normal cell-cycle inhibiting function in many endometrial carcinomas.

MLH1 and MSH2 protein expression as a pre-screening marker in hereditary and non-hereditary endometrial hyperplasia and cancer.

The predictive value of MLH1 or MSH2 protein expression for the presence of truncating germline mutations was examined in benign and (pre)malignant endometrial samples from 3 patient groups: (I) 10 endometrial cancer patients from hereditary non-polyposis colorectal cancer (HNPCC) families with (n = 6) or without (n = 4) a known germline mutation; (II) 15 women from HNPCC families with (n = 7) or without (n = 8) a known germline mutation, who underwent endometrial sampling for non-malignant reasons; (III) 38 endometrial cancer patients <50 years of age, without HNPCC family history. Immunostaining for MLH1 and MSH2 was performed on paraffin-embedded sections. In group III, tumor DNA was examined for microsatellite instability (MSI) and MLH1, MSH2 and MSH6 mutation analysis was carried out. In 6/6 MLH1/MSH2 mutation carriers with endometrial cancer (group I), concordance was found between protein loss in the tumor and the corresponding mutation. In 3 MLH1 mutation carriers, MLH1 protein loss was also observed in concurrent endometrial hyperplasia. In group II, no protein loss was detected in normal endometrial tissue samples; in 3/4 patients with endometrial hyperplasia, MLH1/MSH2 protein loss was observed. In group III, protein loss was detected in 12/38 patients (9 MLH1, 3 MSH2), while in 3/11 patients with concurrent endometrial hyperplasia protein loss was also observed in the hyperplasia. MSI analysis in group III revealed 26 MSI-low and 12 MSI-high tumors. Mutation analysis in 28/38 patients showed only 1 missense MSH6 and no MLH1 or MSH2 germline mutations. In group III, loss of MLH1/MSH2 protein expression was not related to the presence of MSI or MLH1/MSH2 germline mutations. In conclusion, MLH1 or MSH2 protein loss in HNPCC-related endometrial neoplasia is strongly related to corresponding germline mutations. This relation was not clearly present in young sporadic endometrial cancer patients. Immunohistochemical pre-screening of the MLH1 and MSH2 proteins in endometrial hyperplasia or cancer can thus be helpful in HNPCC families. Frequent loss of MLH1 or MSH2 protein in endometrial hyperplasia indicates that this loss is an early event in endometrial carcinogenesis.

Malignancies of the uterine corpus and immunoreactivity score of the DNA "mismatch-repair" enzyme human Mut-S-homologon-2.

We analyzed human Mut-S-Homologon-2 expression in normal endometrial tissue (n = 15) and malignancies of the uterine corpus (n = 40). Human Mut-S-Homologon-2 protein was investigated immunohistochemically on frozen sections, using a highly sensitive streptavidin-peroxidase technique and a specific mouse monoclonal antibody (clone FE11). Human Mut-S-Homologon-2 labeling pattern was compared with the staining pattern of the proliferation marker Ki-67 in the same tumors. A human Mut-S-Homologon-2 immunoreactivity score (human Mut-S-Homologon-2-IRS: negative 0-1; weak 2-3; moderate 4-6; strong 8-12) for semiquantitative analysis of human Mut-S-Homologon-2 expression is presented. In normal endometrial tissue samples we found weak nuclear immunoreactivity for human Mut-S-Homologon-2 in 67%, whereas the remaining 33% were negative for human Mut-S-Homologon-2 (mean human Mut-S-Homologon-2-IRS 1.25 +/- 1.29). All malignancies of the uterine corpus analyzed revealed moderate to strong nuclear immunoreactivity (mean human Mut-S-Homologon-2-IRS 9.00, +/- 3.16). Human Mut-S-Homologon-2 staining was heterogeneous, with visual differences among individual tumor cells. Expression of human Mut-S-Homologon-2 protein was consistently and strongly upregulated in tumor cells of malignancies of the uterine corpus compared with normal endometrial tissue (human Mut-S-Homologon-2-PP p<0.001; human Mut-S-Homologon-2-IS p<0.001; human Mut-S-Homologon-2-IRS p<0.001). No statistically significant correlation in comparing the labeling patterns for human Mut-S-Homologon-2 with the labeling patterns for Ki-67 (mean percentage of Ki-67-positive tumor cells 22.00% +/- 17.20) was observed in malignancies of the uterine corpus (human Mut-S-Homologon-2-PP p=0.443; human Mut-S-Homologon-2-IS p=0.234; human Mut-S-Homologon-2-IRS p=0.173). Our findings indicate that human Mut-S-Homologon-2 is expressed in normal human endometrial tissue and that expression of human Mut-S-Homologon-2 may be of importance for the genetic stability of malignancies of the uterine corpus in vivo. (J Histochem Cytochem 47:113-118, 1999).