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.     

HOXA10 expression in endometrial adenocarcinoma.

Homeobox (HOX) genes are highly evolutionarily conserved regulators of embryonic differentiation. HOX genes are also expressed in normal adult reproductive tissue where they are involved in regulating differentiation. We hypothesized that HOXA10 gene expression may be altered in endometrial adenocarcinoma. To assess HOXA10 gene expression, endometrial tissue was obtained from 32 subjects with normal endometrium or disease confined to the uterus. Nine specimens contained normal endometrium, 5 had endometrial hyperplasia and 18 had stage I and II endometrial adenocarcinoma. RNA was extracted, Northern blot analysis performed and expression of HOXA10 assessed by densitometry. Expression of estrogen receptor, progesterone receptor and p53 was analyzed by immunohistochemistry. HOXA10 was expressed in both normal and neoplastic endometrium. No significant difference in HOXA10 expression was found between normal, hyperplastic and FIGO nuclear grade 1 endometrial tissues. Expression of HOXA10 was increased by 25% in high nuclear grade endometrial adenocarcinomas compared with normal, hyperplastic and low nuclear grade endometrial adenocarcinomas. No difference was noted in expression of estrogen receptor, progesterone receptor or p53. HOXA10 expression is elevated in high nuclear grade stage I and II endometrial adenocarcinomas. Aberrant regulation of HOX gene expression is associated with abnormal differentiation of endometrial tissue.     

Expression of vascular endothelial growth factor (VEGF)-D and its receptor, VEGF receptor 3, as a prognostic factor in endometrial carcinoma.

PURPOSE: To evaluate the prognostic value of vascular endothelial growth factor (VEGF)-D and VEGF receptor (VEGFR)-3 in endometrial carcinoma. EXPERIMENTAL DESIGN: We assessed the levels of immunoreactivity for VEGF-D and VEGFR-3 in 71 endometrial carcinomas, 14 complex atypical endometrial hyperplasias, and 16 normal endometria by immunohistochemistry. RESULTS: VEGF-D was stained in both tumor cells and adjacent stromal cells. VEGFR-3 was stained in both tumor cells and adjacent endothelial cells. Immunoreactivity for VEGF-D in tumor cells and adjacent stromal cells became significantly stronger as lesions progressed from normal endometrium to advanced carcinoma. Similarly, immunoreactivity for VEGFR-3 in tumor cells and adjacent endothelial cells was significantly greater as lesions progressed from normal endometrium to advanced carcinoma. A strong correlation was found between high levels of VEGF-D immunoreactivity in carcinoma cells and VEGFR-3 in both carcinoma cells and adjacent endothelial cells. Similarly, high levels of VEGF-D immunoreactivity in stromal cells were significantly correlated with those of VEGFR-3 in both carcinoma cells and endothelial cells. High levels of VEGF-D in carcinoma cells and stromal cells, as well as those of VEGFR-3 in carcinoma cells and endothelial cells, were significantly related to myometrial invasion and lymph node metastasis. A strong correlation was found between poor survival and high levels of VEGF-D in both carcinoma cells and stromal cells and between poor survival and high levels of VEGFR-3 in carcinoma cells. Moreover, the high levels of VEGF-D in stromal cells and VEGFR-3 in carcinoma cells were independent prognostic factors in endometrial carcinoma. CONCLUSIONS: The presence of VEGF-D and VEGFR-3 in endometrial carcinoma may predict myometrial invasion and lymph node metastasis and may prospectively identify patients who are at increased risk for poor outcome. In addition, VEGF-D and VEGFR-3 may be promising targets for new therapeutic strategies in endometrial carcinoma.     

血型抗原相关性凝集素受体在良恶性宫内膜组织的表达

荆豆素一1,兀鹰素—1—B4受体在增生期子宫内膜腺细胞几为阴性,内膜增生过长时开始出现腺细胞腔缘表达.内膜腺癌时细胞兀鹰素—1—B4在细胞腔缘结合较少或为不规则分布,荆豆素—1受体阳性普遍增强.双花扁豆素受体在增生期和增生过长腺体细胞可呈腔缘,胞浆和基底部混合分布,但腺癌仅见少数腺腔缘阳性细胞.花生素受体表达在不同性质内膜组织间差别不大.以上结果提示子宫内膜腺癌H型和A型血型抗原表达较之增生期和增生过长腺细胞分别有所增减.     

Aromatase expression in stromal cells of endometrioid endometrial cancer correlates with poor survival.

PURPOSE AND EXPERIMENTAL DESIGN: To assess the prognostic significance of intratumoral aromatase in endometrioid endometrial cancer, sections from 55 patients with endometrial cancer were evaluated for expression of aromatase using immunohistochemistry, and the correlation between aromatase expression and clinicopathologic parameters were analyzed. RESULTS: Immunohistochemical staining for aromatase was positive for 32 (58%), 20 (36%), and 19 (34%) patients in cancer epithelial cells, stromal cells, and myometrial cells around the flank invasion, respectively. In situ hybridization also detected aromatase mRNA in all three types of cells. RT-PCR analysis revealed that aromatase mRNA was 2.5 +/- 1.0 amol/mug total RNA (mean +/- SE; n = 7) in tumor tissue. Western blot analysis detected the expected aromatase protein size of 58 kDa in cancer tissues more abundantly than in cancer-free endometrium (n = 3). The immunoreactivity in stromal cells correlated positively with advanced surgical stage and poor survival. Survival analysis revealed that the immunoreactivity of stromal cells was a significant prognostic factor, independent of histologic grade, muscular invasion, and lymph node metastasis, but dependent on surgical stage. By contrast, the immunoreactivity of aromatase both in cancer epithelial cells and myometrial cells did not correlate with prognosis. CONCLUSIONS: To the best of our knowledge, this is the first evidence associating intratumoral aromatase expression in stromal cells and poor survival in endometrioid endometrial cancer. This positive linkage indicates that local expression of aromatase plays a role in tumor progression through the formation of in situ estrogens. In situ expression of aromatase may offer a potential target for management of endometrial cancers.     

Significantly decreased P27 expression in endometrial carcinoma compared to complex hyperplasia with atypia (correlation with p53 expression)

P27 expression was examined on paraffin-embedded specimens in proliferative, secretory, hyperplastic and neoplastic human endometrium by immunohistochemistry. The results of p27 immunoreactivity in endometrial carcinomas were compared with clinicopathological indicators as well as with p53 expression. Thirty-eight cases of endometrial carcinoma, 30 normal functional (15 proliferative, 15 secretory), 24 hyperplastic endometrium (12 without atypia, 12 with atypia) specimens were studied by using monoclonal p27 and p53 antibodies. The streptavidin-biotin-peroxidase detection system was used and the intensity and the distribution of immunoreactivity was evaluated semiquantitatively. p27 expression was present both in the proliferative and secretory phases; the expression being stronger in the secretory period. In complex hyperplasia with atypia, p27 expression was even higher and it was significantly reduced in the endometrial carcinoma group (p<0.05). No significant correlation was found between p27 expression and any of the clinicopathologic prognostic parameters (p>0.05). Nuclear p53 expression was detected in 13 (34.2%) patients with endometrial carcinoma and was higher in non-endometrioid carcinomas and in tumors with increasing FIGO grade (p<0.05). High expression of p53 was not found to be a significant prognostic indicator of survival (p> 0.05). No p53 expression was detected in the endometria with proliferation, secretion or hyperplasia either simple without atypia or complex with atypia. Surprisingly, tumors with absent/low p27 expression showed absent/low p53 expression. Our data suggest that p27 is necessary to control the proliferation of endometrium and its loss of expression seems to play a role in some aspects of endometrial carcinogenesis.     

Expression of the antigenic determinant recognized by the monoclonal antibody 44-3A6 on select human adenocarcinomas and normal human tissues

The IgG1 monoclonal antibody, 44-3A6, was raised against the human lung adenocarcinoma cell line, A549. It has been shown to react with a 40,000 MW protein found on the cell surface, which is preserved in formalin-fixed paraffin-embedded tissues. A recent study of pulmonary carcinomas utilizing immunohistochemical methods showed exclusive binding to lung adenocarcinomas, subsets of neuroendocrine tumors, some carcinoids and a subset of large cell carcinomas. Reactivity was not seen in squamous cell carcinomas and small cell neuroendocrine carcinomas. In addition, melanomas, sarcomas and hematologic malignancies do not express this antigen. We now report on the reactivity pattern of 44-3A6 in adenocarcinomas of nonpulmonary primary sites and in normal adults organs. Strong diffuse staining of neoplastic cells in adenocarcinomas of the stomach, colon, pancreas, gallbladder and breast was noted. Adenocarcinomas arising in the endometrium, ovary, kidney, prostate, thyroid and liver were either negative or showed weak and/or focal reactivity. Strong staining patterns were even noted in adenocarcinomas which had an 'undifferentiated' component; i.e., lacking well-defined glandular elements. Immunoreactivity was noted in epithelial cells in several tissues from which these adenocarcinomas arose including the bronchial tract, stomach, small intestine, pancreas and colon, whereas epithelial cells from the endometrium, kidney, ovary, prostate and thyroid were negative or showed diffuse weak immunoreactivity. Our finding indicate that monoclonal antibody 44-3A6 recognizes an epithelial antigen on subsets of normal as well as transformed glandular epithelia. The differential pattern of expression of its target antigen probably reflects differences in tumor genesis and/or differentiation.     

Neutral endopeptidase 24.11/CD10 suppresses progressive potential in ovarian carcinoma in vitro and in vivo.

Recently, numerous studies have shown that endothelin-1 (ET-1) is expressed in ovarian carcinoma and that ET-1 selectively acts as an autocrine or paracrine growth factor through the endothelin A receptor (ET(A)R), and is involved in cell proliferation, invasiveness, neovascularization, and prevention of apoptosis. Neutral endopeptidase 24.11 (NEP) is a cell surface aminopeptidase with a ubiquitous expression and is capable of degrading a number of bioactive peptides including ET-1. Our previous report showed that stromal NEP expression in ovarian carcinoma was down-regulated as the histologic grade advanced. Here, we confirmed that NEP was expressed in tumor cells as well as stromal tissues in ovarian carcinoma, and investigated the functions of NEP in this carcinoma. We showed that there was a significant decrease in cell proliferation and invasiveness with a reduction in the concentration of ET-1 in the conditioned medium on the NEP overexpression of NEP in ovarian carcinoma cells. In addition, the overexpression of NEP enhanced susceptibility to paclitaxel, resulting in an increased occurrence of apoptotic morphologic change. Furthermore, tumorigenesis was reduced in vivo with the overexpression of NEP, down-regulation of both matrix metalloproteinase-2, and vascular endothelial growth factor expression. This evidence suggests that NEP functionally suppresses the progression of ovarian carcinoma and further study of this enzyme may reveal an effective way to target ET-1 for the treatment of this carcinoma.     

Altered extent, pattern and characteristics of microvascular density are indicators of neoplastic progression in the endometrium

The occurrence, structure and extent of microvascular density were examined in normal endometria, hyperplasia of different types and adenocarcinomas of different degrees of differentiation to determine their biologic and clinical significance in tumor development and progression. Computer-assisted quantitative image analysis was carried out on 12,500 vessels in regard to vessel number, vessel volume, size, shape and extent of vessel antibody staining, with sensitivity and reproducibility exceeding 99%. The results showed the extent, pattern and characteristics of microvascular density to be intimately associated with extent of tumor development and degree of differentiation of the tumor. Vessel number increased with superficial location in normal endometrium, with increased degree of hyperplasia and atypia and with increased degree of dedifferentiation of adenocarcinoma. Increased vessel shape alterations were characteristic of atypical complex hyperplasia when compared to other types of hyperplasia. Vessel number, size and shape were similar in proliferative endometrium and simplex type hyperplasia, and microvascular density in atypical complex hyperplasia was similar to that in well-differentiated adenocarcinoma. The results indicated that vessel shape alterations occur during progression of hyperplasia and vessel size increase occurs in complex-type hyperplasia and in moderately differentiated adenocarcinomas. We conclude that microvascular density is associated with endometrial location and with specific patterns of alteration in different stages of endometrial disease. The results suggest potential clinical applications of vessel analysis for determination of clinical behavior of endometrial preneoplastic and neoplastic alterations.     

Endothelin Converting Enzyme-1 Expression in Endometrial Adenocarcinomas

Endothelin-1 (ET-1) is a potent mitogen in various precursor tumor cells, including endometrial adenocarcinoma. It is proposed that ET-1 produced by endometrial adenocarcinoma may participate in the angiogenesis of this carcinoma in vivo. Endothelin converting enzyme-1 (ECE-1) is the key enzyme that synthesizes ET-1. In this study, we tried to demonstrate the expression of ECE-1 in endometrial carcinomas. Deparaffinized tissue sections from patients with endometrial adenocarcinoma were analyzed by immunohistochemistry for the presence of ECE-1. Our study showed that the expression of ECE-1 was markedly increased in 9 of 15 (60%) well-differentiated endometrial adenocarcinomas; in contrast, only 2 out of 10 (20%) control specimens showed a mild labeling. With new selective inhibitory molecules emerging, research is currently evaluating the possible inhibition of ECE-1 as an alternative approach for the treatment of endometrial as well as other carcinomas.     

芳香化酶P450在子宫内膜癌中的表达及其意义

 目的 探讨芳香化酶P450（P450arom）在正常子宫内膜、增生子宫内膜以及子宫内膜癌中的表达，探讨P450arom在子宫内膜癌发生和发展中的作用。方法 实验分成三组：子宫内膜腺癌组标本50份、子宫内膜增生症组内膜标本27份、正常子宫内膜组标本20份。采用免疫组化（ABC）方法分别测定3组标本中P450arom的表达。结果 P450arom在子宫内膜癌、子宫内膜增生症内膜腺上皮中均阳性表达，两者比较差异无统计学意义（P＞0.05）。P450arom 在正常子宫内膜中无表达。P450arom在子宫内膜腺癌癌组织间质细胞中表达21例（42 %），间质细胞阳性表达与手术病理期别及病理分级密切相关，而与肌层浸润深度、淋巴结转移无关。结论 P450arom在子宫内膜癌发生、发展中起重要作用。子宫内膜腺癌癌组织间质细胞P450arom表达与肿瘤发展及预后密切相关。     

ezrin和flt-1在子宫内膜癌中的表达及其意义

目的探讨膜细胞骨架连接蛋白ezrin、血管内皮生长因子受体1（flt-1）在子宫内膜癌中的表达及其与临床病理参数之间的关系.方法采用免疫组化方法检测79例子宫内膜癌和12例转移灶组织中ezrin和flt-1蛋白的表达,选择不典型增生14例,单纯、复合性增生20例和正常子宫内膜增生期15例作为对照.结果 ezrin蛋白在正常内膜,单纯、复合性增生, 不典型增生和内膜癌四组间的差异有显著性（P＜0.01）,不典型增生组ezrin的表达高于单纯、复合性增生,内膜癌高于不典型增生.多数正常和增生腺上皮细胞不表达ezrin,而大多数不典型增生和内膜癌细胞表达ezrin,且为细胞浆和细胞膜弥散着色,差异有显著性.内膜癌中,深肌层浸润组ezrin的表达高于浅肌层或无肌层浸润组（P＜0.05）.flt-1在不典型增生中的表达高于正常内膜,内膜癌中flt-1的表达强度随组织学分级升高而降低（P＜0.05）.内膜癌中ezrin与flt-1的表达间无明显相关性,ezrin与雌激素受体（ER）间存在负相关.结论 ezrin可能参与了子宫内膜癌的发生和演进过程.     

Expression and prognostic significance of urokinase and plasminogen activator inhibitor type-1 in endometrial hyperplasia and cancer

Proteolytic enzymes, like urokinase (uPA) and plasminogen activator inhibitor type-1 (PAI-1), are involved in remodelling tissues during invasion and metastasis of tumor cells. The purpose of the study is to evaluate the expression and the prognostic significance of these enzymes in endometrial hyperplasia and cancer. We used immunohistochemical staining to localize uPA and PAI-1 antigens and evaluate their expression, and the enzyme-linked immunosorbent assay (ELISA) to measure their levels during the progression of endometrial carcinoma. The results show that the levels of uPA and PAI-1 detection are systematically weak in simplex hyperplasia and are moderate in complex hyperplasia. In the endometrial carcinoma a very strong reaction was observed in the most aggressive variant of epithelial tumors. A positive signal for uPA was found only in the cytoplasm of normal and hyperplastic cells while, in tumors, uPA was present also in the cellular areas surrounding the neoplastic glands and at the apex of the malignant cells. The PAI-1 immunoreactivity was weak to moderate in 95.4% of carcinomas, with a diffuse signal mostly distributed in the cytoplasm of neoplastic cells and tumor stroma. UPA antigen concentrations were significantly higher in endometrial carcinoma than in endometrial hyperplasia (p<0.05) and in normal endometrium (p<0.001). PAI-1 antigen concentrations in carcinoma samples were significantly higher than in normal endometrium (p=0.002), but the difference was not statistically significant with respect to that in endometrial hyperplasia. We did not find any correlation between uPA and PAI-1 concentrations and the standard prognostic parameters for evaluating endometrial carcinoma. In conclusion, this study demonstrates that in hyperplastic endometria and in endometrial carcinoma there is a progressive increase in expression of uPA and PAI-1 than in normal endometrial tissue. In carcinoma tissues, the high expression of uPA is unregulated in the surrounding stroma tissue, particularly in the most aggressive histopathologic variants. UPA and PAI-1 may be factors associated with invasive behavior in endometrial carcinoma independent of other clinicopathological parameters.     

Defining the extent of cables loss in endometrial cancer subtypes and its effectiveness as an inhibitor of cell proliferation in malignant endometrial cells in vitro and in vivo

Loss of Cables expression is associated with a high incidence of endometrial hyperplasia and endometrial adenocarcinoma in humans. The Cables mutant mouse develops endometrial hyperplasia and following exposure to chronic estrogen develops early endometrial adenocarcinoma. The objectives of the current study were to determine if: (1) loss of Cables expression occurred in high grade endometrioid adenocarcinoma, uterine serous and clear cell carcinoma as observed in endometrial hyperplasia and low grade endometrial adenocarcinoma; (2) overexpression of Cables inhibited cell proliferation in endometrial cancer (EC) cells in vitro and in vivo; and (3) progesterone could regulate the expression of Cables mRNA. Hyperplastic endometrium and low and high grade endometrioid adenocarcinoma showed loss of Cables expression when compared to benign control secretory endometrium. Loss of Cables expression in serous and clear cell tumors was similar to that observed in endometrioid adenocarcinomas with greater than 80% showing loss of protein expression. Treatment of EC lines with progesterone increased cables expression in low-grade EC whereas it had no effect on cables expression in cells derived from high-grade EC. The progesterone-induced increase in cables was abrogated in the presence of a progesterone receptor (PR) antagonist, suggesting the PR mediates the increase. Cables overexpression inhibited cell proliferation of well differentiated EC cells and had no effect on the poorly differentiated EC cells. The capacity to form tumors was dramatically reduced in the Cables overexpressing cell lines compared to those cells containing the control vector. Collectively these results suggest that Cables is an important regulator of cell proliferation and loss of Cables expression contributes to the development of all types of EC.     

Expression of vasohibin as a novel endothelium-derived angiogenesis inhibitor in endometrial cancer

We have previously reported on vasohibin as a novel endothelium-derived vascular endothelial growth factor (VEGF)–inducible inhibitor of angiogenesis. The aim of our present study was to define the role of vasohibin in endometrioid endometrial adenocarcinoma. We collected 78 sections of endometrial carcinoma for assessment using immunohistochemistry. Twenty-seven were well differentiated (G1), 25 were moderately differentiated (G2), and 26 were poorly differentiated endometrioid adenocarcinomas (G3). We also included 12 sections of normal cyclic endometria, six of which were in the proliferative phase and six were in the secretory phase. We investigated the expression of vasohibin, and compared it to VEGF receptor-2 (VEGFR-2: KDR/flk-1), CD34, Ki-67, VEGF-A, and D2-40 (as a lymphatic vessel marker). We assessed the ratio of vasohibin- and VEGFR-2-positive vessels in the stroma of endometrial carcinoma. Immunohistochemical assessment was classified as negative or positive based on staining intensity. Vasohibin was selectively expressed on vascular endothelial cells in both cyclic endometria and endometrial carcinomas. Vasohibin was highly expressed in the normal functional endmetrium of the secretory phase, especially in the spiral artery, and was highly expressed in all grades of endometrioid adenocarcinomas. The stromal endothelial cells in G3 expressed vasohibin and VEGFR-2 more frequently than these in G1. In endometrioid adenocarcinomas, there was a significant correlation between the expression percentage of vasohibin and that of VEGFR-2 ( P <  0.0001, r ² = 0.591). This is the first study to elucidate the correlation between expression of vasohibin in the stromal endothelial cells and that of VEGFR-2 in human carcinomas. ( Cancer Sci 2008; 99: 914–919)