Galectin-3 expression in normal,hyperplastic, and neoplastic endometrial tissues

This study evaluated the expression of galectin-3 in 101 curettage specimens from normal, hyperplastic, and neoplastic endometrial tissues using immunohistochemistry. The histologic diagnoses were as follows: normal proliferative (n = 8) and secretory (n = 4) phase, simple hyperplasia (SH, n = 16), complex hyperplasia without atypia (CH, n = 11), atypical hyperplasia (AH, n = 13), endometrioid adenocarcinoma (EC, n = 35), serous papillary carcinoma (SPC, n = 10), and clear cell carcinoma (CC, n = 4). Immunostaining was scored with regard to the approximate percentage of positive tumor cells and relative staining intensity. The scores of immunostaining increased significantly from NE, SH, CH, and AH to the adenocarcinomas (ANOVA, p < 0.0001). Subsequently, three significantly different levels of galectin-3 expression were found (Newman-Keuls multiple comparison test). These consisted of (a) NE, SH, and CH, (b) AH and EC, and (c) SPC and CC. Galectin-3 expression increased with tumor grade (ANOVA, p = 0.0026). The scores of FIGO stages I to III did not differ significantly (ANOVA, p = 0.1687). Enhanced nuclear galectin-3 expression was noted in carcinomas, immunostaining of stromal cells decreased in the latter. This study shows that galectin-3 expression increases from normal and hyperplastic to atypical hyperplastic and cancerous states of endometrial tissues, and provides further evidence of a relationship between AH and EC.     

Distribution of estrogen receptors in various cell types of normal, hyperplastic, and neoplastic human endometrial tissues

To facilitate our understanding of estrogen participating in the genesis of human endometrial hyperplasia and carcinoma and the influence of the surrounding mesenchyme in their development, we assayed estrogen receptors in the epithelium and stroma of various human endometrial tissues by immunocytochemistry. The receptors were localized in fresh frozen sections using the ER-ICA kits (Abbott Laboratories, North Chicago, Illinois). The receptor content was highest in the proliferative endometrium and decreased gradually throughout the postovulatory phase of the menstrual cycle in both the epithelium and the stroma and this occurred more rapidly in the stroma. Estrogen receptor content was high in both the epithelium and the stroma of hyperplasia and was thus similar to that of the proliferative endometrium, the receptor content was low in the epithelium of endometrial intraepithelial neoplasia (hyperplasia with cytologic atypia) closely resembling that of carcinoma. In carcinomas there was a heterogenous distribution of estrogen receptors both in the epithelium and the stroma, but in general, the receptor content was low as compared with normal proliferative or hyperplastic endometrium; however, in selected cases the content of estrogen receptor in the stroma was high, despite the lack of receptors in the epithelium.     

Expression of survivin in normal, hyperplastic, and neoplastic colonic mucosa

The regulation of apoptotic cell death may have a profound effect on the pathogenesis and progression of colon cancer. Survivin, a member of the inhibitor of apoptosis gene family, has been detected in fetal tissue and in a variety of human malignancies. In the current study, we investigated survivin expression by an immunohistochemical approach in benign, hyperplastic, premalignant, and malignant lesions of the colon. Survivin was detected in all cases of normal colonic mucosa (20/20), hyperplastic polyps (20/20), adenomatous polyps (20/20), and in both well differentiated and moderately differentiated colonic adenocarcinomas (20/20). In the normal colonic mucosa, survivin expression was mostly restricted to the base of the colonic crypts. All epithelial cells showed uniformly intense staining for survivin in hyperplastic polyps. By contrast, adenomas and adenocarcinomas showed a heterogeneous staining pattern with cell-to-cell, gland-to-gland, and regional variability in the intensity of survivin staining. In contrast to the basal preponderance of staining in normal colonic mucosa, numerous survivin positive cells were present at the luminal surface of hyperplastic polyps, adenomatous polyps, and adenocarcinomas. In conclusion, the expression of survivin is not a specific marker of adenocarcinoma of the colon but does show characteristic and reproducible patterns of expression in non-neoplastic proliferative lesions and in normal colonic mucosa.     

Tenascin expression in normal, hyperplastic, dysplastic and neoplastic canine mammary tissues

Mammary tumours are the most common neoplasias of female dogs and may have a complex histological pattern with both epithelial and spindle cells participating in the transformation process. A frequent feature of these tumours is chondroid or bone metaplasia of the extracellular matrix, which mainly occurs in areas of proliferated spindle-shaped cells, probably of myoepithelial origin. The present study evaluates immunohistochemically the expression of tenascin in 186 surgical samples of canine mammary tissues, ranging from normality to neoplasia. Tenascin was present in all mammary tissues studied, with an increased expression in remodelling situations and in neoplastic lesions. Basement membrane was the most frequently labelled structure, but stromal tissue was more often and widely labelled in neoplastic lesions. The extracellular matrix was positive in solid and anaplastic carcinomas as well as in spindle cell proliferation areas. Tenascin expression in extracellular matrix was also abundant in areas of initial chondroid metaplasia and, with variable extension, in almost all cartilage islands of mixed tumours. In well differentiated secretory areas only apical granules of luminal cells were positive, suggesting a different pattern of tenascin expression during secretory differentiation. The digestion of chondroitin sulphate significantly improved the labelling for tenascin when a co-expression of these two molecules was present. Although our results suggest that tenascin cannot be used as a marker of transformation or of malignancy in canine mammary oncology, it is clear that this molecule plays an important role in proliferation and differentiation processes in the canine mammary gland.     

Argyrophil cells in normal, hyperplastic, and neoplastic endometrium.

Scanty argyrophil cells are present in a substantial proportion of normal endometria, particularly during the secretory stage of the cycle. Argyrophil cells are also present in the various types of hyperplastic endometria and are found in more than half of endometrial adenocarcinomas. In some endometrial neoplasms they are present in abundance, but tumours rich in such cells do not have any features suggestive of a carcinoid tumour and are morphologically identical to adenocarcinomas of similar grade which are devoid of argyrophil cells. Endometrial adenocarcinomas containing argyrophil cells tend to be well differentiated and tend not to invade deeply into the myometrium. It is suggested that Müllerian epithelial stem cells possess a potentiality for differentiation into APUD cells.     

Expression of KAI1 in paraffin-embedded normal, hyperplastic and neoplastic prostate and prostate carcinoma cell lines

Expression of KAI1, a tumor metastasis suppressor gene, was studied with different fixatives in frozen and paraffin-embedded sections of human and rat prostate carcinoma cell lines and human prostate lesions by Immunohisto-chemistry. Immunoreactivity of the membrane antigen in cell lines was associated with known expression levels in these lines and the fixative used. Formalin and paraformaldehyde helped maintain the immunoreactivity of cells. In human prostate, frozen sections revealed diffuse reactivity of the antigen in normal and neoplastic tissues while paraffin-embedded tissues usually showed focal reactivity, although more than 50% of cases with normal epithelium and adenocarcinomas were reactive. In some cases, pretreatment with trypsln enhanced immunoreactivity. Benign prostatic hyperplasia (BPH) showed the most intense diffuse immunoreactivity, which suggested enhanced expression. Prostatic intraepithelial neoplasia (PIN) also often expressed high levels of KAI1. Three of five metastases were reactive but two primaries and their metastases were not. Lymphocytes in primary carcinomas and lymphocytes and germinal center cells in lymph nodes were immunoreactive, while adjacent primary or metastatic prostate adenocarcinoma epithelium was not immunoreactive. Although paraffin-embedded human tissues were not optimal for determining levels of expression of KAI1, they did show immunoreactivity that could have prognostic value and showed the specific cytoplasmlc localization of the protein in cells.     

Nucleolar organizer regions in normal,hyperplastic and neoplastic parathyroid glands

Summary Using a one-stage silver staining technique, nucleolar organizer regions (AgNORs) were studied in paraffin sections of parathyroid glands (and in two lymph node metastases) from patients operated upon because of hyperparathyroidism or thyroid disease. The parathyroids were microscopically differentiated into normal, hyperplastic, adenomatous and carcinomatous glands. AgNORs were observed as distinct black dots of varying size and somewhat varying configuration in the nuclei of all glands. The mean number of AgNORs in the hyperplastic and adenomatous glands was not significantly different from that in the normal glands, whereas the carcinomatous glands exhibited significantly increased mean AgNOR number. No evidence was obtained for a role of AgNOR counting in the differentiation between normal and hyperplastic or adenomatous parathyroids, but the results suggest a potential role of enumeration of AgNORs in the discrimination between benign and malignant parathyroid neoplasms.     

Poly(ADP-ribose) polymerase (PARP) and DNA-fragmentation factor (DFF45): expression and correlation in normal, hyperplastic and neoplastic endometrial tissues

This study evaluated the immunohistochemical expression of poly(ADP-ribose) polymerase (PARP) and DNA fragmentation factor 45 (DFF45) in normal endometria (NE, n=13), non-atypical (NAH, n=22) and atypical hyperplasia (AH, n=14), endometrioid carcinoma (EC, n=34), serous carcinoma (SC, n=10), and clear cell carcinoma (CCC, n=2). With regard to quantity and intensity of positively stained cells, immunostaining was scored as negative, low, and strong. Nuclear PARP immunoreactivity was found in all cases. If present, DFF45 immunoreactivity was detected predominantly in the nucleus and to some extent in the cytoplasm. PARP immunoreactivity increased significantly from NE via NAH to AH (P=0.0004), and decreased from AH to endometrial carcinomas (P=0.0054). DFF45 immunoreactivity increased significantly from NE to NAH and to AH (P=0.0009). No significant differences were calculated between AH and endometrial carcinomas (P=0.7495). FIGO stages and tumor grades could not be characterized by PARP and DFF45 immunoexpression (P=1.000 and 0.7383, as well as P=0.3034 and 0.7533, respectively). Pearson correlation revealed significant associations of PARP and DFF45 immunoscores for all diagnostic categories of NE, NAH, AH, EC, and SC/CCC. Immunoexpression of PARP and DFF45 is apparently altered in endometrial carcinomas as compared with non-neoplastic endometrial tissues, indicating impaired mechanisms of apoptosis in the former.     

Expression of parathyroidlike protein in normal, proliferative, and neoplastic human breast tissues.

Parathyroidlike peptide (PLP), or parathyroid hormone-related protein, is a protein of uncertain biological function that is structurally homologous to parathyroid hormone. Immunohistochemical studies have identified aminoterminal epitopes of PLP in breast carcinomas, but not in normal breast. In the present studies, immunohistochemistry and in situ hybridization were performed to evaluate further expression of PLP in normal, proliferative, and neoplastic breast tissues. Using a polyclonal antibody that recognizes epitopes within the middle and carboxyl-terminal domains of PLP, immunoreactive protein was detected within the cytoplasm of lobular and ductal epithelial cells in all normal and fibrocystic breast tissues from 74 patients. The intensity of cytoplasmic staining was increased in association with lactation, adenosis, and simple or atypical ductal hyperplasia and decreased in atrophic lobules. Cytoplasmic reactivity was also observed in 69% (56 of 81) of breast adenocarcinomas. Expression of immunoreactive PLP was inversely correlated with tumor stage and extent of nodal involvement at the time of diagnosis. However, there was no significant correlation with tumor grade, patient age, or hormone receptor status. In situ hybridization studies confirmed the epithelial expression of PLP messenger RNA in PLP-positive normal and neoplastic breast tissue. Interestingly, tumor-associated calcifications were identified in 43% of PLP-positive carcinomas, but in only 12% of PLP-negative carcinomas (P < 0.007). Our results suggest that PLP plays some role in the normal differentiated function of mammary epithelial cells and are consistent with the hypothesis that expression of this protein influences local calcium metabolism.     

The proapoptotic factor HLDF in the normal, hyperplastic and neoplastic endometrium

Antibodies to the factor HLDF are shown to be specific markers of apoptosis and permit the estimation of the rate of programmed cell death in the course of a normal menstrual cycle and in pathologic endometrial processes. HLDF expression in the epitheliocytic cytoplasm makes it possible to evaluate apoptosis at early stages, before the emergence of the first morphological signs and after apoptotic body formation. The study shows increased apoptotic processes at the end of a normal menstrual cycle and during neoplastic cell transformation. Antibodies to the HLDF factor may be used as a new immunohistochemical marker for the differential diagnosis of benign and malignant endometrial processes.     

Expression of the nm23 homologues nm23-H4, nm23-H6, and nm23-H7 in human gastric and colon cancer

Eight members of the nm23-gene family have been described. The involvement of nm23-H1 and nm23-H2 in tumour progression and metastasis, as well as in gene regulation and apoptosis, has been shown in numerous studies. Whether nm23-H4, -H6, and -H7 play a role in tumours is, however, largely unknown. This study describes data on the expression of these three nm23 homologues in human colon and gastric cancer by real-time RT-PCR and immunohistochemistry. Increased expression of these genes, most strikingly nm23-H4 and -H7, was observed in the majority of tumours analysed. No correlation with tumour stage according to the TNM classification was found. In contrast, by immunohistochemical analysis, nm23-H4 and -H6 overexpression correlated with the intestinal tumour type in gastric cancer tissues, whereas no increased immunoreactivity for the three nm23 proteins was noted in the diffuse type tumour specimens. These findings indicate that nm23-H6, and particularly nm23-H4 and -H7, may be involved in the development of colon and gastric carcinoma, the latter possibly in a type-specific manner. A contribution to tumour progression or metastasis could not, however, be proven. Elucidation of the specific mechanisms by which the nm23 homologues nm23-H4, -H6, and -H7 are involved in tumour development requires further studies.     

Basement membranes in fetal,adult normal,hyperplastic and neoplastic human prostate

Summary The distribution of the various basement membrane (BM) components (type IV collagen, laminin and heparan sulphate proteoglycan) was studied in fetal, adult normal, hyperplastic and neoplastic prostates in formalin- and ethanol-fixed paraffin-embedded specimens. Stromal, epithelial and neoplastic BMs expressed differential susceptibility to pepsin treatment, suggesting conformational differences in the expression of epitopes on BM proteins in distinct anatomical structures and various lesions of the human prostate. In fetal prostate the acinar BM was regular and continuous in contrast to normal adult prostate and various hyperplastic conditions where the acinar BM was locally thickened or unreactive to the anti-BM antibodies. The localization pattern of BM components in grade I and grade II phases of prostatic cancer did not differ essentially from those found in various hyperplastic lesions. Regardless of the histopathological grade of malignancy, prostatic carcinoma cells were surrounded by distinct pericellular and periacinar membranes which were present even at points of contact with the stroma. This suggests that stroma invasion is invariably associated with neoplastic BM formations. Immunohistochemical evidence of the stromal or epithelial origin of neoplastic BMs could not be found. However, the consistent extracellular distribution of neoplastic BM components in contact with the stroma indicates that the elaboration of BM material requires a stromal influence.     

Silver-stained nucleolar organizer regions in the normal,hyperplastic and neoplastic endometrium

Summary A morphometric evaluation of number and grouping of megakaryocytes (MK) in five different groups of chronic myeloproliferative disorders (CMPD) was performed by counting 60 high power fields equaling approximately 14.28 mm² of haematopoiesis in each case. Twenty-one up to 29 cases were evaluated for each of five categories of CMPD and one control group; a total of 132 cases of CMPD and 33 control cases were used. The mean number of MK per square millimetre was 15.54±1.53 in chronic myeloid leukaemia of common or granulocytic type (CML.CT), 69.91±5.85 in CML with megakaryocytic increase (CML.MI), 59.59 ±3.27 in polycythaemia vera (P. vera), 59.85±4.59 in primary thrombocythaemia (PTH), 67.58±4.11 in chronic megakaryocytic granulocytic myelosis (CMGM), and 19.7±3.07 in controls. The distinction between free or isolated MK, and between clustered or grouped MK corresponds to the total cell counts of MK in the various groups of CMPD. Clustering of MK was significantly higher in CMGM and PTH compared to other groups, but the difference between them was not statistically significant. Significant differences in the mean number of MK were obtained between controls and CML.CT on the one hand and all other groups of CMPD on the other. The results further support the histological sub-classification of CMPD according to the primary disorders of the Hannover classification (not advanced by sclerosis, fibrosis or excess of blasts, respectively).     

Differential distribution of tenascin in the normal, hyperplastic, and neoplastic breast.

We studied by immunohistochemistry the distribution of tenascin with the monoclonal antibody 100EB2, and compared it with that of laminin in breast tissue samples from fetal, adult resting, lactating, and aging parenchyma, variants of fibrocystic disease, fibroadenomas, cystosarcoma phylloides, and ductal and lobular carcinomas. Monoclonal antibodies were applied to cryosections by the avidin-biotin-complex method; selected samples were studied by double immunofluorescence, and by Western blot analysis. In adult resting and aging breasts, tenascin immunoreactivity was noted in the periductal and periacinar stromal regions as thin irregular bands; in the lactating breast, broader periductal bands were observed. In these samples, laminin immunoreactivity was a single continuous line around ducts, acini, and vessels. In fetal breasts, tenascin appeared as thick periductal bands, whereas laminin remained as a delicate single line. In FCD, tenascin increased around ducts showing hyperplasia, papillomas and apocrine metaplasia, whereas laminin retained its delicate linear pattern. Similar patterns were seen in fibroadenomas and cystosarcoma phylloides with variable tenascin reactivity in the stroma beyond the ducts. Tenascin immunoreactivity was markedly increased around ducts containing in situ carcinoma appearing as broad bands, whereas that of laminin showed a linear, frequently discontinuous appearance. Prominent stromal tenascin immunoreactivity was seen in infiltrating ductal and lobular carcinomas, whereas laminin was virtually absent save for scattered lines. The abundance of tenascin in the carcinomatous stroma contrasted with its scarcity in the non-neoplastic stromal regions. By Western blotting, both chains of tenascin with molecular weights 250,000 and 180,000 were shown in ductal and lobular carcinomas as well as in normal breast. Tenascin immunoreactivity was noted in the periepithelial stromal regions of adult resting and aging breast ducts and acini. The amount of tenascin was moderately enhanced in certain physiologic conditions (fetal growth, gestation), as well as hyperplasias, dysplasias (fibrocystic disease) and benign tumors, whereas it was markedly enhanced in intraductal and infiltrating carcinomas. During fetal mammary development, adult physiologic and pathologic hyperplasias, and in carcinomas, the increasing tenascin reactivity contrasted with the stable or decreasing laminin reactivity.     

Adrenomedullin, Bcl-2 and microvessel density in normal, hyperplastic and neoplastic endometrium

Adrenomedullin (ADM) is a multifunctional 52-amino acid peptide involved in numerous physiological and pathological processes, including angiogenesis, growth regulation, differentiation, and vasodilation. ADM is thought to act through the G protein-coupled receptor calcitonin receptor-like receptor, with specificity being conferred by receptor-associated modifying protein 2. The aim of the present study was to clarify the roles of ADM status, and tumor vessels in endometrium. Specimens were examined for ADM, microvessel density (MVD), area of venules (AV) and Bcl-2 oncoprotein using an immunoperoxidase method. The difference of ADM between normal proliferative phase and hyperplasia without atypia was significant ( P < 0.05). The level of Bcl-2 was significantly different between hyperplasia without atypia and hyperplasia with atypia ( P < 0.05). ADM, MVD and AV in the endometrium increased in a stepwise manner from normal, simple or complex hyperplasia with or without atypia to grade 1 adenocarcinoma. In contrast, expression of Bcl-2 oncoprotein was decreased. These parameters identify the role of ADM expression and Bcl-2 protein in relation to cell growth and vasodilating in the neoplastic changes.     

Expression of Birt-Hogg-Dubé gene mRNA in normal and neoplastic human tissues.

Birt-Hogg-Dubé (BHD) syndrome is an inherited autosomal genodermatosis characterized by hamartomas of the hair follicle called fibrofolliculomas and an increased risk for developing spontaneous pneumothorax, lung cysts and renal neoplasia. BHD was localized to chromosome 17p11.2 by linkage analysis in BHD families, and germline insertion/deletion and nonsense mutations in a novel gene were identified which were predicted to prematurely truncate the BHD protein, folliculin. No homology to other human proteins was found although folliculin was conserved across species. As a first step toward understanding the function of BHD in the cell and how BHD mutations can lead to the BHD phenotype, we measured the expression of BHD mRNA in normal and neoplastic human tissues by fluorescent in situ hybridization. BHD mRNA was expressed in a variety of tissues, including the skin and its appendages, the distal nephron of the kidney, stromal cells and type 1 pneumocytes of the lung, acinar cells of the pancreas and parotid gland, and epithelial ducts of the breast and prostate. In the brain, BHD mRNA was expressed in neurons of the cerebrum, and Purkinje cells in the cerebellum. BHD mRNA was also expressed in macrophage and lymphocytes in the tonsils and spleen. Tissues with reduced expression of BHD mRNA included heart, muscle and liver. BHD mRNA was expressed strongly in the proliferating epithelial strands of fibrofolliculomas, the cutaneous lesions characteristic of BHD, but not in renal tumors from BHD patients. These results indicate a wide expression pattern for BHD mRNA in many tissues, including skin, lung and kidney, which are involved in the BHD phenotype, and support a tumor suppressor role for BHD in renal cancer.     

DNA-flow-cytometric measurements on the normal,atrophic, hyperplastic and neoplastic human endometrium

Summary DNA distribution patterns and the fractions of the cell cycle phases were determined by means of flow-through cytometry in 87 samples of normal, atrophic, hyperplastic and carcinomatous human endometrium. The S-phase fractions vary during the normal menstrual cycle between 1 and 3% and reach a periovulatory maximum between 4.4 and 4.7%. Atrophic endometrium and regressive-glandular cystic hyperplasia have little DNA synthesis (1.01% and 1.68% S-phase fractions respectively). Proliferating glandular cystic hyperplasia reveals 3.38% S-phase fraction, whereas adenomatous hyperplasia has an increased number of DNA-synthesizing cells (4.81%). The well-differentiated endometrial carcinoma shows no cytophotometrically detectable differences in comparison to adenomatous hyperplasia. All endometrial samples except for poorly differentiated endometrial carcinoma showed a diploid to tetraploid DNA distribution pattern. The poorly differentiated endometrial carcinoma displays two different types: one rapidly growing diploid-tetraploid tumor with 8.0 to 9.6% S-phase fractions, and another type with stemline deviations, polyploid nuclei and less pronounced synthetic activity.Dedicated to Professor Volker Becker at the occasion of his 60th anniversary