E-cadherin Immunostaining Research Articles

Parity is associated with lower cervical E‐cadherin expression in postmenopausal women

Epithelial cadherin (E-cadherin), a transmembrane glycoprotein involved in calcium-dependent homophilic cell-cell adhesion, is expressed aberrantly during cervical carcinogenesis. E-cadherin expression and putatively implicated predictors in healthy women remain a rather under-investigated area. The objective of this study is to evaluate the possible associations between E-cadherin expression and reproductive/lifestyle factors in cervical epithelial cells from postmenopausal women. A total of 105 healthy postmenopausal women (aged 45-68 years old) attending a university menopause clinic were enrolled in this cross-sectional study. Pap smears were derived and E-cadherin immunostaining was evaluated in squamous, glandular and squamous metaplastic cells, using a semi-quantitative method (rating scale: 0-3). Reproductive and lifestyle factors were obtained from patients' chart review. In squamous cells, women with a history of 0-1 deliveries presented with a higher score vs women with 2-4 deliveries (P = 0.003). Social drinkers and women drinking alcohol daily exhibited a higher E-cadherin immunostaining score in squamous cells vs non-drinkers (0.96 +/- 0.72 vs 0.56 +/- 0.65, P = 0.004). A higher dietary calcium intake was marginally correlated with a lower staining score in squamous cells (0.94 +/- 0.78 for low, 0.71 +/- 0.70 for average, 0.45 +/- 0.52 for high consumption, P = 0.073). E-cadherin expression seems to be associated with reproductive history and lifestyle habits in squamous cervical cells from healthy postmenopausal women. E-cadherin might participate in the molecular mechanisms underlying the role of parity as a risk factor for cervical cancer.

Interobserver variability and aberrant E-cadherin immunostaining of lobular neoplasia and infiltrating lobular carcinoma

The distinction between lobular neoplasia and infiltrating lobular carcinoma from ductal neoplasia and infiltrating duct carcinoma with equivocal histologic features may present a challenge as this distinction has important therapeutic implications. Although E-cadherin staining has been of value in helping to make this determination, the variability of the E-cadherin staining pattern and the immunohistochemistry techniques can be problematic in clinical practice. A total of 161 cases of breast lesions previously diagnosed as lobular neoplasia and infiltrating lobular carcinoma were selected from the departmental files. Three surgical pathologists interpreted them in a blinded manner for the histology diagnoses and E-cadherin staining. E-cadherin staining was conducted on the paraffin-embedded sections of the breast lesions using two different source antibodies. Our results using morphology and E-cadherin stain agreed with the previous diagnoses of lobular neoplasia and infiltrating lobular carcinoma in 140 of 161 cases (86.9%). Among the 140 cases, three pathologists agreed with the morphologic diagnoses of lobular neoplasia and infiltrating lobular carcinoma in 100 (71.4%), two pathologists in 26 (18.6%) and one pathologist in 14 (10%). All three pathologists disagreed with the previous diagnoses of lobular neoplasia and infiltrating lobular carcinoma but reevaluated as ductal lesions in 21 cases (13.0%). E-cadherin staining was confirmatory in 136 of total 161 cases (84.5%) of both lobular and duct lesions by showing the loss of staining in lobular lesions and the presence of complete membrane staining in duct lesions. Aberrant E-cadherin reactions were retained weak or partial incomplete thin membrane reaction in lobular-type lesions and reduced membrane reaction in ductal-type lesions were seen in 25 of the total 161 cases (15.5%). E-cadherin immunoreaction with two different antibodies showed discrepant results in 5 of 78 cases tested (6.4%). This study illustrates (1) interobserver variability of the morphologic diagnoses of lobular neoplasia/infiltrating lobular carcinoma and duct neoplasia/infiltrating duct carcinoma, (2) the occasional presence of aberrant E-cadherin stain pattern in these breast lesions and (3) variability of E-cadherin immunostaining results by two different antibodies.

Cyclin D1 expression analysis in familial breast cancers may discriminate BRCAX from BRCA2-linked cases

Most familial breast cancers arise in patients who tested negative for germline mutations in BRCA1 and BRCA2 genes (also referred to as BRCAX cases). Several studies aimed to define histopathological and molecular profiles characteristic of BRCA1, BRCA2 and BRCAX tumors have been performed. Major pathological and immunohistochemical differences have been reported in BRCA1 cancers compared to the other two groups, whereas less difference has been observed between BRCA2 and BRCAX cases. The aim of this study was to investigate the ability of selected tumor markers to discriminate BRCAX breast cancers from cancers arising in carriers of mutations in BRCA genes, and their usefulness in selecting familial cases in whom testing for such mutations is more likely to result uninformative. We carried out a morphological and immunohistochemical analysis on 22 BRCA1, 16 BRCA2 and 33 BRCAX familial breast cancers. Age at first diagnosis, histological type and grade, and immunostaining for estrogen receptor (ER), progesterone receptor (PR), p53, HER2/Neu, E-cadherin and cyclin D1 were investigated. The occurrence of somatic mutations of the TP53 gene was also verified. BRCA1 tumors resulted clearly distinguishable from BRCAX cases, occurring at a younger age, being more frequently of higher grade, negative for ER, PR and cyclin D1 expression and positive for p53 alterations. The predictive value of age at diagnosis, histological grade and PR expression was confirmed in a multivariable analysis. When comparing BRCA2 with BRCAX tumors, the only parameter that differed was cyclin D1, which was significantly overexpressed in BRCA2 cases both in the univariable and the multivariable analyses. If confirmed by further studies, our observations indicate that the investigation of cyclin D1 expression in familial breast cancer cases could be used, in conjunction with the analysis of other tumor markers preferentially associated with BRCA1 or BRCA2 tumors, to prioritize hereditary cases for mutation testing in BRCA genes.

E-cadherin but not β-catenin expression is decreased in laryngeal biopsies from patients with laryngopharyngeal reflux

Abnormal exposure of acid refluxate on the esophageal mucosa has been shown to decrease the epithelial barrier function through an alteration in the intercellular junctional complex. However, only few studies have examined the molecular effects caused by abnormal exposure of gastric refluxate on the laryngeal epithelium. E-cadherin and beta-catenin are cell membrane-associated proteins playing a major role in the maintenance of cell-cell adhesion in epithelial tissues. In this study we tried to analyse the molecular effect of laryngopharyngeal reflux (LPR) on the cellular expression of these proteins. Therefore, we compared the expression of E-caherin and beta-catenin in laryngeal biopsies from patients with and without pH-documented laryngopharyngeal reflux. Paraffin-embedded archival laryngeal biopsies taken from 21 patients, who had undergone rigid laryngoscopy under general anaesthesia and postoperative 24-h pH monitoring, were evaluated immunohistochemically with antibodies to E-cadherin and beta-catenin. The membrane expression of the two proteins was categorized in no expression, mild, moderate and strong (grade 0-3). In LPR patients (n=14) the mean grade of E-cadherin and beta-catenin expression was 1.57 and 1.21, while in specimens of patients without pH-documented LPR it was 2.57 and 1.29. The difference in E-cadherin expression was statistically significant (P=0.011). From our findings we conclude that LPR can cause a decrease in the laryngeal expression of E-cadherin but not of beta-catenin. The reduction of E-cadherin-mediated adhesion could contribute to the development of laryngeal neoplasms. E-cadherin immunostaining of laryngeal biopsies could be a further diagnostic tool to confirm the diagnosis in patients with suspected LPR.

The effect of hyaluronic acid size and concentration on branching morphogenesis and tubule differentiation in developing kidney culture systems: Potential applications to engineering of renal tissues

Hyaluronic acid (HA) is a glycosaminoglycan of tissue engineering importance that plays a vital role in mammalian development. In vitro kidney culture methods were utilized to investigate the importance of HA during renal organogenesis. We found that HA has the ability to simultaneously modulate ureteric bud (UB) branching, promote mesenchymal-to-epithelial transformation, and promote differentiation of both metanephric mesenchyme (MM) and the UB depending on the concentration and molecular weight (MW) of HA. Hyaluronidase inhibited branching morphogenesis in both isolated UB and whole kidney cultures, suggesting endogenous HA is required for branching morphogenesis. HA exhibited morphogen-like properties, stimulating branching morphogenesis at low concentrations (0.1%) and low MW (6.55 kDa), but inhibiting at high concentrations (3.75%) and high MW (234.4 kDa). Furthermore, HA of every MW tested promoted collecting duct differentiation as measured by AQP-2 expression. E-cadherin immunostaining and qPCR of nephron differentiation markers (OAT-1, NaP i-2, AQP-1, and THP) demonstrated that HA of a variety of MWs strongly promotes mesenchymal epithelialization and nephron differentiation in a concentration-dependent manner. Since the HA synthesis and degradation genes, has-2 and hyal-2, are highly expressed during kidney development, this data suggests that specific sizes and concentrations of HA may act to independently regulate UB branching and promote tubular maturation, representing a potential switch for ending branching morphogenesis, as well as initiating nephron differentiation. In addition, the ability of HA to promote in vitro embryonic kidney growth and maturation, together with the biocompatibility and crosslinking capability of HA, suggests a potential use of HA for both creating an instructive, 3D scaffold for in vitro kidney engineering from developmental tissues, as well as promoting tubule regeneration in injured or cryopreserved kidneys.

E-cadherin expression in cervical epithelial cells of postmenopausal women: association with hormone therapy, tibolone, and raloxifene

This study assesses the possible associations between postmenopausal therapy (hormone therapy, raloxifene, and tibolone) and E-cadherin expression in normal cervical Papanicolaou smears (squamous, glandular, and metaplastic cells). E-cadherin immunostaining was less intense in metaplastic cells of women on tibolone, whereas hormone therapy and raloxifene were not associated with altered E-cadherin expression.

Loss of cell-adhesion molecule complexes in solid pseudopapillary tumor of pancreas

Solid pseudopapillary tumor of pancreas (SPT) is a rare neoplasm that occurs most often in young females with the two distinct features, the ‘solid-cystic' gross appearance, and the ‘solid-pseudopapillary' microscopic pattern. It has been reported that almost all SPT tumors contain a mutation in the β-catenin gene; however, the histogenetic origin of this tumor remains largely a mystery. E-cadherin is a cell adhesion molecule that links to catenins to form cell adhesion junctions, which is associated with the cytoskeleton formation. In this study, we examined the expression of E-cadherin and β-catenin from SPT in an attempt to determine the molecular basis for the unusual morphology of this tumor. Nine cases of SPT were retrieved from Surgical Pathologic Archives of three institutions, including one male and eight females. H&E slides of each case were reviewed to confirm the diagnosis. The β-catenin gene was sequenced in one case. E-cadherin and β-catenin immunostains, were performed on all nine cases. Sequencing analysis on one case showed a point mutation of the β-catenin gene, confirming previous findings that almost all SPT tumors contain mutation in the β-catenin gene. Immunostains showed that, in both solid and pseudopapillary areas, all the tumor cells lost expression of E-cadherin, and β-catenin nuclear expression was observed in all cases. Our findings suggest that loss of cytoplasmic β-catenin protein in the cell adhesion complex due to β-catenin gene mutation, results in instability of the complex, loss of E-cadherin in cell membrane, and eventually dissociation of the tumor cells to form the pseudopapillary pattern.

E-cadherin expression and bromodeoxyuridine incorporation during development of ovarian inclusion cysts in age-matched breeder and incessantly ovulated CD-1 mice

BackgroundFemale CD-1/Swiss Webster mice subjected to incessant ovulation for 8 months and 12-month breeder mice both developed ovarian inclusion cysts similar to serous cystadenomas. The majority of cysts appeared to be dilated rete ovarii tubules, but high ovulation number resulted in more cortical inclusion cysts. We hypothesized that comparison of inclusion cyst pathology in animals of the same age, but with differences in total lifetime ovulation number, might allow us to determine distinguishing characteristics of the two types of cyst.MethodsOvaries from breeder mice (BR) or females subjected to incessant ovulation (IO) were compared at 6-, 9- and 12-months of age. Ovaries were serially sectioned and cysts characterized with regard to location and histology, E-cadherin immunoreactivity and rates of BrdU incorporation.ResultsInclusion cysts developed with age in BR and IO ovaries. The majority of cysts were connected to the ovarian hilus. Two cortical inclusion cysts were observed in ten IO ovaries and one in ten BR ovaries. Low or no E-cadherin immuno-staining was seen in the OSE of all mice studied. Conversely, strong membrane immuno-staining was observed in rete ovarii epithelial cells. Variable E-cadherin immunoreactivity was seen in cells of hilar inclusion cysts, with strong staining observed in cuboidal ciliated cells and little or no staining in flat epithelial cells. Two of the three cortical cysts contained papillae, which showed E-cadherin immuno-staining at the edge of cells. However hilar and cortical cysts were not distinguishable by morphology, cell type or E-cadherin immunoreactivity. BrdU incorporation in cyst cells (1.4% [95% CI: 1.0 to 2.1]) was greater than in OSE (0.7% [95% CI: 0.4 to 1.2]) and very few BrdU-labeled cells were observed in rete ovarii at any age. Incessant ovulation significantly increased BrdU incorporation in OSE of older animals.ConclusionThese experiments confirm ovarian inclusion cysts develop with age in the CD-1 mouse strain, irrespective of total ovulation burden. We conclude longer periods of incessant ovulation do not lead to significant changes in inclusion cyst formation or steroidogenesis in CD-1 mice and inclusion cyst type can not be distinguished by morphology, cell proliferation rate or E-cadherin immunoreactivity.

Isolation of Mouse Pancreatic Ductal Progenitor Cells Expressing CD133 and c-Met by Flow Cytometric Cell Sorting

Islet transplantation has become available across the globe since a novel protocol was reported. However, because donors are in short supply, only a minority of patients benefit from this procedure. Pancreatic progenitor cells are a promising resource for regeneration of new islets, but whether progenitor cells reside in ductal epithelium is not clear. Mouse pancreas was examined by immunohistochemistry with cell surface markers specific for ductal cells. We developed an isolation method for ductal cells by flow cytometric cell sorting using a newly identified specific marker for ductal cells. By using an in vitro colony assay, we characterized their proliferative and multipotent capacity. CD133 is expressed specifically in ductal epithelium. Flow cytometric analysis revealed that purified ductal cells are highly enriched in the CD133(+)CD34(-)CD45(-)Ter119(-) fraction. An analysis of clonal epithelial colonies formed by individual cells revealed that progenitor cells with multilineage differentiation capacity are present in neonatal ductal epithelium. Moreover, these progenitor cells express c-Met. In adult mice, progenitor cells that show a high proliferative capacity but appear committed to a ductal lineage are co-purified with CD133(+)CD34(-)CD45(-)Ter119(-) cells. We established a system for isolating and culturing mouse pancreatic ductal cells that relies on flow cytometric cell sorting. Clonal analysis revealed that a population of progenitor cells is present among CD133(+) ductal cells. Isolation of these cells will facilitate future studies into the roles of pancreatic progenitor cells in regeneration and carcinogenesis.

Characterization of columnar cell lesions of the breast: immunophenotypic analysis of columnar alteration of lobules with prominent apical snouts and secretions

Columnar cell lesions of the breast are detected with increasing frequency in routine pathology practice, in part as a result of the widespread biopsy of nonpalpable breast abnormalities detected by screening mammography. Immunohistochemical investigation of the lesions in relation to the normal breast or to other breast pathologies is not well characterized, and the malignant potential of this spectrum of lesions has not been examined clinically. In this study, a cohort of 45 breast specimens containing columnar cell lesions, in particular, columnar alteration of lobules with prominent apical snouts and secretions (CAPSS), was investigated for expression of a series of breast tumor biomarkers. Using a semiquantitative immunohistochemical scoring system, up-regulation of estrogen, progesterone, and androgen receptors in CAPSS lesions to levels not significantly different from that in in situ or invasive breast tumors was identified. In four cases where CAPSS within a specimen lacked expression of a steroid hormone receptor, the coexisting in situ or invasive carcinoma also lacked expression of that receptor. In 81% of CAPSS lesions, E-cadherin immunostaining was reduced in isolated foci of cells or was decreased in intensity in all cells within the lesion. Quantitation of Ki-67 immunostaining demonstrated that proliferation of cells within CAPSS lesions was increased, compared with normal breast epithelium, but was lower than that detected in in situ or invasive cancers within the same specimens. Results of these analyses indicate that CAPSS shares immunophenotypic alterations with other premalignant lesions, the clinical implications of which may be investigated using established breast tumor biomarkers.

Reduced structural proteins in the conjunctival epithelium in allergic eye disease

Allergic eye disease affects up to 20% of the population with varying severity. The conjunctival epithelium plays a key role in allergic eye disease. The purpose of this study was to determine whether the conjunctival epithelium is abnormal in allergic eye disease. Conjunctival biopsy samples were taken from patients with seasonal allergic conjunctivitis (SAC) 'in' and 'out of season' and nonatopic control subjects. Specimens were fixed in glycol methacrylate, 2 microm serial sections cut and Image-J used to assess the sites and areas of immuno-staining. E-cadherin, CD44, keratins K5/6, K8, K13, K14, K18 and pan-keratin immuno-staining were all significantly lower in patients 'out of season' compared with normal controls. No structural differences in the epithelium were observed between the two groups. The epithelium of patients 'in season' was thicker and immuno-staining of the above markers similar to controls. The expression of a wide spectrum of epithelial cell adhesion proteins and cytoskeletal elements is downregulated in the conjunctiva of SAC patients 'out of season' compared with normal controls. We suggest that this could have an important impact on the ability of the epithelium to protect itself against allergen penetration, potentially influencing the development and course of allergic eye disease and offering a novel area for therapeutic control.

Chemopreventive alteration of the cell-cell adhesion in head and neck squamous cell cancer

Approximately 310,000 new cases of oral and pharynx cancer account for a major cause of neoplasm related morbidity and mortality world-wide. Unfortunately, the survival rate has not improved significantly in the last decade. The vast majority of head and neck cancer is squamous cell carcinoma. The major adhesion-proteins involved in the development and maintenance of all solid tissue are the Cadherins. Cadherins are the transmembrane components of the adherent junction with interaction with plakoglobin and beta-catenin. Downregulation of Cadherins and catenins is frequently observed in many types of human cancer. Sulindac sulfone is one of the new therapeutic apoptotic agents that show promise in the treatment of cancer. In this study, we incubated sulindac sulfone with a head and neck cancer cell line and investigated the outcome of E-Cadherin. Immunohistochemical and Western blot analyses were then performed, with different concentrations of sulindac sulfone (100, 200, 400, 600, and 800 microMol) for 48 h. At 400 microMol of sulindac sulfone a decrease of 21% was observed; at 600 microMol, 44% decrease of beta-catenin concentration was seen, and incubation with 800 microMol resulted in 73% reduction of secreted beta-catenin. Incubation with sulindac sulfone seemed to stop proliferation; however, with respect to the controls, there was no increased reduction of the total protein. Sulindac sulfone resulted in an increase of E-Cadherin content in the head and neck squamous cell cancer cell line after 48 h of incubation; however, the reactivity was restricted to the adherent junctions. At increasing concentrations of sulindac sulfone, intercellular E-Cadherin immunostaining intensifyied. ELISA also depicted significant rising levels of E-Cadherin. Sulindac sulfone contributes to the inactivation of cGMP phospho-diesterase. Thus, the accumulation of cellular cGMP and protein kinase G is induced. The following degradation of the phosphorylated beta-catenin and the dissociation from the Cadherin-catenin complex releases E-Cadherin. This may also contribute to growth inhibition and co-ordinate with apoptosis induction. It is not really clear as to, which pathway results in the elevation of the E-Cadherin proteins. However, in epithelial cancer cells, the Cadherin-catenin complex serves as a target for the chemopreventive agent, sulindac sulfone.

Aberrant E-cadherin staining patterns in invasive mammary carcinoma

BackgroundE-cadherin, a cell surface protein involved in cell adhesion, is present in normal breast epithelium, benign breast lesions, and in breast carcinoma. Alterations in the gene CDH1 on chromosome 16q22 are associated with changes in E-cadherin protein expression and function. Inactivation of E-cadherin in lobular carcinomas and certain diffuse gastric carcinomas may play a role in the dispersed, discohesive "single cell" growth patterns seen in these tumors. The molecular "signature" of mammary lobular carcinomas is the loss of E-cadherin protein expression as evidenced by immunohistochemistry, whereas ductal carcinomas are typically E-cadherin positive.Patients and methodsWe report on E-cadherin immunostaining patterns in five cases of invasive mammary carcinomaResultsThese were five exceptional instances in which the E-cadherin immunophenotype did not correspond to the apparent histologic classification of the lesion. These cases which are exceedingly rare in our experience are the subject of this report.ConclusionFindings such as those illustrated in this study occur in virtually all biologic phenomena and they do not invalidate the very high degree of correlation between the expression of E-cadherin and the classification of breast carcinomas as ductal or lobular type on the basis of conventional histologic criteria.

E-cadherin immunoreactivity correlates with recurrence and progression of minimally invasive transitional cell carcinomas of the urinary bladder

E-cadherin is a transmembrane glycoprotein involved in intercellular adhesion. Abnormal (i.e., lost or decreased) expression of E-cadherin has been linked to invasiveness of many malignant tumors, including bladder carcinomas. To our knowledge, studies analyzing the prognostic impact of E-cadherin immunoreactivity especially in minimally invasive transitional cell bladder carcinomas (stage pT1) have not been published in the Anglo-American literature. In the present study, we immunostained 69 cases of pT1 transitional cell bladder carcinomas for E-cadherin using multitissue arrays. The results were compared with p53 and Ki-67 antigen immunoreactivity, clinicopathological parameters and the patients' outcome. E-cadherin immunoreactivity, which was found abnormal in 42% of cases, correlated significantly with substage (pT1a/pT1b; p=0.029) and p53 index (p=0.041) and tendentiously with Ki-67 antigen index (p=0.089) and age (p=0.07). By univariate Cox regression analysis, abnormal E-cadherin immunostaining correlated significantly (p=0.005) with early tumor recurrence, but not with early tumor progression (p=0.168). In a multivariate analysis, this parameter was identified, besides tumor grade (p=0.002), as an independent predictor of recurrence-free survival (p=0.016). Concerning tumor progression, age was identified as the single independent prognostic parameter (p=0.041), but E-cadherin immunoreactivity displayed a tendentious independent predictive value in this respect (p=0.071). We conclude from our data that immunohistochemical E-cadherin staining may provide additional prognostic information in patients with pT1 bladder carcinomas.

Prognostic significance of dysadherin expression in patients with non–small cell lung cancer

The aim of this study was to evaluate the expression of dysadherin and E-cadherin and to investigate their clinical significance as prognostic factors in non-small cell lung cancer. Non-small cell lung cancer specimens were obtained from 131 patients undergoing clinically indicated operations at the Department of General and Cardiothoracic Surgery, Kanazawa University Hospital, between 1995 and 1997. All patients had undergone curative resection of the primary tumor, including systematic lymph node dissection. The avidin-biotin-peroxidase complex method was used for immunostaining of dysadherin and E-cadherin. Among the 131 lung cancer specimens, 46 (35.1%) tumors were positively stained with dysadherin. Preserved membranous E-cadherin staining was present in 45.8% (60/131) of cases. In this analysis dysadherin expression was not correlated with E-cadherin expression (P = .1333), but a significant association was observed between dysadherin expression and survival time. The overall survival of patients with dysadherin-positive tumors was significantly worse than that of those with dysadherin-negative tumors (P = .0059). Patients with reduced E-cadherin immunopositivity survived significantly shorter than those with preserved E-cadherin immunopositivity (P = .0406). The overall survival of patients with positive dysadherin and reduced E-cadherin expression was significantly worse than that of patients with negative dysadherin and preserved E-cadherin expression (P = .0002). Multivariate analysis revealed the independent prognostic value of dysadherin positivity, reduced E-cadherin expression, and lymph node metastasis on overall survival. Dysadherin expression is an independent prognostic factor of survival in patients with non-small cell lung cancer, and combined immunohistochemical analysis of dysadherin and E-cadherin expression might provide further prognostic information.

Expression of Connexins 26 and 43 in Canine Hyperplastic and Neoplastic Mammary Glands

Gap junctions are the only communicating junctions found in animal tissues and are composed of proteins known as connexins. Alterations in connexin expression have been associated with oncogenesis; reported studies in rodent and human mammary glands, which normally express connexins 26 and 43, confirm these alterations in malignancies. Mammary neoplasms represent the second most frequent neoplasm in dogs, and since there are no reports on the study of connexins in canine mammary glands, the present study investigated the expression of connexins 26 and 43 in normal, hyperplastic, and neoplastic mammary glands of this species, to verify if altered patterns of connexin staining are related to higher cell proliferation and malignant phenotypes. A total of 4 normal, 8 hyperplastic mammary glands, 9 benign, and 51 malignant mammary gland neoplasms were submitted for the immunostaining of connexins 26 and 43, E-cadherin, and proliferating cell nuclear antigen (PCNA). Normal, hyperplastic, and benign neoplastic mammary glands showed a punctate pattern for connexin 26 and 43 staining and an intercellular E-cadherin staining. Malignant neoplasms, especially the most aggressive cases with high cell proliferation rates, presented either fewer gap junction spots on the cell membranes or increased cytoplasmic immunostaining. Malignant tumors also expressed a less intense immunostaining of E-cadherin; the expression of this adhesion molecule is important for the transportation of connexins to cell membranes and in forming communicating gap junctions. Deficient expression of E-cadherin could be related to the aberrant connexin localization and may contribute to the malignant phenotype. In conclusion, the expression and distribution of connexins and E-cadherin are inversely correlated to cell proliferation in malignant mammary neoplasms of dogs and may well be related to their more aggressive histologic type and biologic behavior.

FTY720, a fungus metabolite, inhibits in vivo growth of androgen‐independent prostate cancer

FTY720, a derivative of fungus, has demonstrated dramatic anticancer effect in several malignancies recently. Our study evaluates the therapeutic potential of FTY720 in the treatment of androgen-independent prostate cancer using a human prostate cancer xenograft in nude mice. CWR22R, an androgen-independent human prostate tumor xenograft was inoculated into castrated nude mice and the animals were administrated with either normal saline or FTY720 (10 mg/kg) through intraperitoneal (i.p.) injection for 20 days. Body weight and tumor volume were recorded every 2 days, and serum prostate specific antigen (PSA) levels were also measured before and after the treatment. The effect of FTY720 on tumor cell proliferation was examined using antibodies against PCNA and Ki-67 by immunohistochemical staining, MTT assay and colony forming assay, whereas apoptotic effect of FTY720 was evaluated by TUNEL assay and immunostaining using antibodies against cleaved caspase 3 and Bcl-2. In addition, the potential inhibitory effect of FTY720 on prostate cancer angiogenesis and metastasis was investigated by immunostaining of CD31, VEGF, E-cadherin and beta-catenin. Our results showed that FTY720 treatment led to suppression of CWR22R tumor growth without causing any detectable side effects in nude mice. The FTY720-induced tumor suppression was correlated with decreased serum PSA level as well as reduced proliferation rate, suppression of angiogenic factors, and restoration of E-cadherin and beta-catenin expression. In addition, the FTY720-treated tumors showed increased apoptosis rate demonstrated by increased TUNEL- and cleaved caspase 3-positive cells, and decreased Bcl-2 expression. Our results suggest a potential novel agent in the suppression of androgen-independent prostate cancer.

Transcriptome analysis in blastocyst hatching by cDNA microarray*

Hatching is an important process for early embryo development, differentiation and implantation. However, little is known about its regulatory mechanisms. By integrating the technologies of RNA amplification and cDNA microarrays, it has become possible to study the gene expression profile at this critical stage. Pre-hatched and hatched ICR mouse embryos (25 blastocysts in each group were used in the triplicate experiments) were collected for RNA extraction, amplification, and microarray analysis (the mouse cDNA microarray, 6144 genes, including expressed sequence tags). According to cDNA microarray data, we have identified 85 genes that were expressed at a higher level in hatched blastocyst than in pre-hatched blastocysts. In this study, 47 hatching-related candidate genes were verified via re-sequencing. Some of these genes have been selected and confirmed by real-time quantitative RT-PCR. These hatching-specific genes were also expressed at a lower level in the delayed growth embryos (morula or blastocyst without hatching at day 6 post hCG). These genes included: cell adhesion and migration molecules [E-cadherin, neuronal cell adhesion molecule (NCAM), lectin, galactose binding, soluble 7 (Lgals7), vanin 3 and biglycan], epigenetic regulators (Dnmt1, and SIN3 yeast homolog A), stress response regulators (heme oxygenase 1) and immunoresponse regulators [interleukin (IL)-2-inducible T-cell kinase, IL-4R, interferon-gamma receptor 2, and neurotrophin]. The immunostaining of E-cadherin and NCAM showed strong and specific localization in hatched blastocyst. This work provides important information for studying the mechanisms of blastocyst hatching and implantation. These hatching-specific genes may have potential as new drug targets for controlling fertility.

Pleomorphic variant of invasive lobular carcinoma of the male breast

Dear Editors: Lobular carcinoma of the male breast is extremely rare, with approximately 20 reported cases in the literature, all of the classic type [2, 4]. Herein, we describe a case of pleomorphic lobular carcinoma occurring in a male breast, with documentation of E-cadherin immunostaining. A 44-year-old Ashkenazi (Eastern European) Jewish man, with three children, presented with a left breast mass of approximately 4 months duration. There was no history of trauma, gynecomastia, liver disease, estrogen administration, or drug use. The patient’s family history was significant in that both of his grandmothers died of breast carcinoma. Physical examination revealed a firm mass localized beneath the nipple. There were no palpable axillary lymph nodes. Excisional biopsies of the breast mass were followed by mastectomy with axillary lymph node dissection. The specimen consisted of a firm, gray-white mass with poorly circumscribed margins, measuring 2.5×2.0 cm. Microscopic sections showed an infiltrating tumor composed of discohesive pleomorphic tumor cells. Few ducts were seen, one of which showed ductal carcinoma in situ, solid type, intermediate grade (Fig. 1). The tumor cells infiltrated in a classic single linear pattern with a concentric targetoid pattern around neoplastic and non-neoplastic ducts. Individual tumor cells were large, with eccentric nuclei with irregular nuclear borders and some enlarged nucleoli (Fig. 2). The cell cytoplasm was frequently eosinophilic and vacuolated with intracytoplasmic lumina (signetring cell appearance). Occasional multinucleated cells were observed. Few mitoses were seen. Neither necrosis nor calcifications were found. There was no evidence of gynecomastia. The nipple was normal. There were 13 axillary lymph nodes without metastases. Immunohistochemical studies demonstrated positive membrane staining for E-cadherin in the non-neoplastic ducts and in the intraductal carcinomatous component and complete loss of E-cadherin expression in the invasive component (Fig. 3). The Ki-67 proliferation index was positive in 5% of the tumor cells. Staining for gross cystic disease fluid protein (GCDFP-15)—the apocrine differentiation marker—was negative. The tumor cells were found to be positive for both estrogen and progesterone receptors and negative for Her-2/neu protein. Subsequent genetic screening for BRCA (tumor suppressor gene) mutations was negative in this patient. The diagnosis of pleomorphic invasive lobular carcinoma, grade 3, was made. The patient was treated with radiotherapy and chemotherapy and was alive and well, without evidence of recurrent or metastatic disease, 2 years after surgery. Carcinomas of the male breast, grossly and microscopically, are remarkably similar to those seen in females. As such, they can be in situ or invasive [5]. The predominant histological type in males, in all large series, has been infiltrating duct carcinoma, with scattered reports of infiltrating lobular carcinoma, all of the classical type, characterized by small, round, regular cells, arranged in single lines, with a targetoid pattern of infiltration. Pleomorphic lobular carcinoma, which was defined in 1992 in the female breast, has a poorer prognosis than its classic counterpart [1]. This subtype demonstrates an infiltrative pattern identical to that of classic lobular carcinoma; however, the nuclei show greater pleomorphism, increased chromatin clumping, increased mitotic activity, single or multiple prominent nucleoli, and, usually, abundant cytoplasm. The degree of nuclear atypia may approach that which is found in infiltrating duct carcinoma, but the invasive pattern characteristic of the classic lobular variant is always well maintained. Immunohistochemical staining for E-cadherin may help in the differential diagnosis. E-cadherin is an epitheliumspecific molecule involved in cell-to-cell adhesion. As such, B. Maly (*) . A. Maly . K. Meir . O. Pappo Department of Pathology, Hadassah University Hospital, Kiryat Hadassah, p.o.b.12000, 91120 Jerusalem, Israel e-mail: Maly_Bella@ yahoo.com Fax: +972-2-6426268

Composite glandular-endocrine cell carcinoma of the stomach. Report of two cases with goblet cell carcinoid component

Composite glandular-endocrine cell carcinoma (CGECC) is recognized as a special type of gastric tumor composed of ordinary adenocarcinoma and neuroendocrine tumors. Goblet cell carcinoid (GCC) is a well-established type of appendiceal carcinoid, but the GCC component has not been well delineated in CGECC of the stomach. We report on two gastric CGECCs with a GCC component, analyzing the histologic components by immunohistochemistry. On initial biopsy, both cases were diagnosed as signet-ring cell carcinoma. However, the resected tumors consisted of three components: signet-ring cell carcinoma, GCC, and glandular adenocarcinoma. Although some signet-ring carcinoma cells and goblet carcinoid cells were indistinguishable by hematoxylin and eosin staining, E-cadherin immunostaining disclosed a definitive difference regarding the staining pattern in these cells. Both patients are well, with no recurrent tumor for about 10 years of follow-up. CGECC with a GCC component may have been confused with conventional adenocarcinoma with signet-ring cells. In cases of advanced signet-ring cell carcinoma with good prognosis, the possibility of such CGECC has to be considered.