E‐cadherin immunohistochemistry in breast pathology: uses and pitfalls

E‐cadherin immunohistochemistry is used commonly in surgical pathology practice to help distinguish lobular carcinoma in situ from ductal carcinoma in situ and invasive lobular carcinoma from invasive ductal carcinoma in histologically problematic or indeterminate cases. However, the interpretation of E‐cadherin immunostains is not always straightforward. Failure to recognize the pitfalls and limitations of E‐cadherin immunostains can lead to an erroneous diagnosis which may result in inappropriate patient management, particularly for patients with in‐situ lesions. In this paper we review the uses and, particularly, the pitfalls in the interpretation of E‐cadherin immunostains in distinguishing lobular from ductal lesions of the breast.     

Interaction of KLRG1 with E‐cadherin: New functional and structural insights

The killer cell lectin‐like receptor G1 (KLRG1) is an inhibitory receptor expressed by memory T cells and NK cells in man and mice. It is frequently used as a cell differentiation marker and members of the cadherin family are ligands for KLRG1. The present study provides new insights into the interaction of mouse KLRG1 with E‐cadherin. Firstly, we demonstrate that co‐engagement of KLRG1 and CD3/TCR in a spatially linked manner was required for inhibition arguing against the notion that KLRG1‐ligation per se transmits inhibitory signals. Secondly, experiments with T cells carrying Y₇F‐mutant KLRG1 molecules with a replacement of the tyrosine residue to phenylalanine in the single ITIM indicated that the inhibitory activity of KLRG1 is counteracted to some degree by increased interaction of KLRG1⁺ T cells with E‐cadherin expressing target cells. Thirdly, we demonstrate that deletion of the first or the second external domain of E‐cadherin abolished reactivity in KLRG1‐reporter cell assays. Finally, we made the intriguing observation that KLRG1 formed multimeric protein complexes in T cells in addition to the previously described mono‐ and dimeric molecules.     

P‐cadherin functional role is dependent on E‐cadherin cellular context: a proof of concept using the breast cancer model

P‐cadherin overexpression is associated with worse breast cancer survival, being a poor prognostic marker as well as a putative therapeutic target for the aggressive triple‐negative and basal‐like carcinomas (TNBCs). Previously, we have shown that P‐cadherin promotes breast cancer invasion of cells where membrane E‐cadherin was maintained; however, it suppresses invasion in models without endogenous cadherins, like melanomas. Here, we investigated if P‐cadherin expression would interfere with the normal adhesion complex and which were the cellular/molecular consequences, constituting, in this way, a new mechanism by which E‐cadherin invasive‐suppressor function was disrupted. Using breast TNBC models, we demonstrated, for the first time, that P‐cadherin co‐localizes with E‐cadherin, promoting cell invasion due to the disruption caused in the interaction between E‐cadherin and cytoplasmic catenins. P‐cadherin also induces cell migration and survival, modifying the expression profile of cells expressing wild‐type E‐cadherin and contributing to alter their cellular behaviour. Additionally, E‐ and P‐cadherin co‐expressing cells significantly enhanced in vivo tumour growth, compared with cells expressing only E‐ or only P‐cadherin. Finally, we still found that co‐expression of both molecules was significantly correlated with high‐grade breast carcinomas, biologically aggressive, and with poor patient survival, being a strong prognostic factor in this disease. Our results show a role for E‐ and P‐cadherin co‐expression in breast cancer progression and highlight the potential benefit of targeting P‐cadherin in the aggressive tumours expressing high levels of this protein. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

E‐cadherin: From epithelial glue to immunological regulator

E‐cadherin is best known as a central molecule in adherens junctions, joining adjacent epithelial cells together, thereby safeguarding epithelial barrier function. However, recent findings have uncovered an immunological role for this adhesion molecule, linked to its expression in dendritic cells (DCs) and alternatively activated macrophages (MPHs) and its impact on intracellular signaling pathways. In this respect, E‐cadherin has been shown to influence the immunogenicity/tolerogenicity of DCs through the regulation of β‐catenin functionality. For Langerhans cells (LCs), the DC type found in the skin epidermis, E‐cadherin is known to mediate interactions with keratinocytes (KCs), thereby immobilizing immature LCs in the epidermis and preventing their maturation. In this issue of the European Journal of Immunology, a study by Mayumi et al. [Eur. J. Immunol. 2013. 43: 270–280] now describes a role for E‐cadherin in the final steps of LC differentiation from human peripheral blood monocytes. Although TGF‐β induces LC‐like cells, these intermediates still express the dermal DC marker DC‐SIGN along with Langerin; E‐cadherin ligation is sufficient to induce the full LC phenotype in these cells. Here, we place these findings in the context of current knowledge and propose new avenues for future research.     

Immunohistochemical analysis of the expression of E‐cadherin and ZEB1 in non‐small cell lung cancer

We performed an immunohistochemical analysis of the expression of zinc‐finger E‐box binding homeobox 1 (ZEB1), a master regulator of epithelial‐mesenchymal transition (EMT), and determined its relationship with E‐cadherin in 157 non‐small cell lung carcinomas (93 adenocarcinomas, 36 squamous cell carcinomas, 18 large cell carcinomas, and 10 pleomorphic carcinomas). Although the expression of E‐cadherin was low in the subset of adenocarcinomas (10%) and squamous cell carcinomas (11%), ZEB1 expression was only observed in one case of squamous cell carcinoma and none of the adenocarcinomas. In contrast, the low expression of E‐cadherin (50% and 90%, respectively) and the positive expression of ZEB1 (11% and 50%, respectively) were more frequently observed in poorly differentiated carcinomas (large cell carcinomas and pleomorphic carcinomas). Overall, the expression of ZEB1 was inversely correlated with that of E‐cadherin. Furthermore, the distribution of ZEB1‐positive cancer cells was more restricted than in the area in which the expression of E‐cadherin was lost, and the former was detected within the latter. We concluded that the expression of ZEB1 was not necessarily associated with the low expression of E‐cadherin in lung adenocarcinomas and squamous cell carcinomas. The expression of ZEB1 correlated with an undifferentiated and/or sarcomatoid morphology that may occur in the late stage of EMT.     

Prognostic significance of single isolated cells with decreased E‐cadherin expression in pseudomyxoma peritonei

Pseudomyxoma peritonei (PMP) cases can be classified into the prognosis‐related subtypes of disseminated peritoneal adenomucinosis (DPAM) and peritoneal mucinous carcinomatosis (PMCA). To investigate the mechanisms of mucinous invasion and the differing prognoses of these two subtypes, we examined the expression levels of proteins involved in cellular adhesion and invasion, including E‐cadherin, vimentin, β‐catenin, and S100A4, in single isolated tumor cells (SICs) and cohesive cellular strips within mucin pools isolated from DPAM (n = 31) and PMCA (n = 21) patients. In both PMCA and DPAM cases, SICs showed a complete loss of E‐cadherin expression, whereas cells in cohesive cellular clusters retained E‐cadherin expression. The frequency of high numbers of SICs (>8) in PMCA cases was significantly greater than that in DPAM cases (86% and 26%, respectively) and was correlated with poor progression‐free survival (P = 0.019) in a univariate analysis. In both PMP subtypes, strong vimentin expression was identified in most of the SICs but not the cohesive cellular strips. The relatively slow progression of DPAM may be attributable to the smaller number of SICs that lack E‐cadherin expression and have increased vimentin expression, whereas the rapid progression of PMCA may be due to larger numbers of these SICs.     

Slug regulates E‐cadherin expression in metastatic adenocarcinoma cells isolated from pleural fluid

Slug protein is a key regulator of epithelial‐mesenchymal transition, but its expression in cancer is less well studied. To evaluate the expression of slug, E‐cadherin and vimentin in adenocarcinoma cells from malignant pleural effusions, we analyzed 121 malignant pleural fluid specimens. Twenty‐eight nonmalignant pleural fluid specimens were analyzed as control. Besides clinical cytological diagnosis tests, immunofluorescence, immunocytochemistry and Western blotting methods were used. Results showed strong membrane staining of E‐cadherin in adenocarcinoma cells from pleural fluid. Slug mainly showed nucleus staining. Cytoplasma positive of vimentin was found in adenocarcinoma cells isolated from pleural fluid. Slug, E‐cadherin and vimentin expression was found in 43/121 (36%), 87/121 (72%) and 102/121 (84%) cases, respectively. Our data showed elevated levels of slug were accompanied by down regulation of E‐cadherin and the expression of vimentin in adenocarcinoma cells. In addition, there was no relationship between slug expression and patient's age or gender or tumor site. Hyperplasia epithelium cells from nonmalignant pleural fluid were uniformly negative for E‐cadherin and slug. In conclusion, the results demonstrated the inverse expression of slug and E‐cadherin in the majority of malignant pleural fluid cases compared with nonmalignant pleural fluid. The slug protein may be helpful to access the prognosis of patients with pleural fluid. Diagn. Cytopathol. 2013. © 2011 Wiley Periodicals, Inc.     

E‐cadherin germline mutations define an inherited cancer syndrome dominated by diffuse gastric cancer

To extend earlier observations of germline E‐cadherin mutations in kindreds with an inherited susceptibility to diffuse gastric cancer, we searched for germline E‐cadherin mutations in five further families affected predominantly by diffuse gastric cancer and one family with a history of diffuse gastric cancer and early‐onset breast cancer. Heterozygous inactivating mutations were found in the E‐cadherin gene in each of these families. No mutation hotspots were identified. These results demonstrate that germline mutation of the E‐cadherin gene is a common cause of hereditary diffuse gastric cancer and suggest a role for these mutations in the incidence of breast cancer. Hum Mutat 14:249–255, 1999. © 1999 Wiley‐Liss, Inc.     

Polymorphonuclear neutrophils promote dyshesion of tumor cells and elastase‐mediated degradation of E‐cadherin in pancreatic tumors

Pancreatic ductal adenocarcinoma (PDAC) presenting with a micropapillary growth pattern is frequently associated with a prominent neutrophil infiltration into the tumor. The relevance of neutrophil infiltrates for tumor progression, however, is still debated. To gain insight into the role of polymorphonuclear neutrophils (PMNs) in PDAC, we assessed their effect on pancreatic tumor cells grown in vitro as monolayers. Time‐lapse video microscopy showed a PMN‐induced dyshesion of the tumor cells, and subsequent experiments revealed that this dyshesion was due to PMN elastase‐mediated degradation of E‐cadherin, an adhesion molecule that mediates the intercellular contact of the tumor cells. E‐cadherin degradation by elastase or — (for comparison) down‐modulation by specific siRNA, significantly increased the migratory capacity of the pancreatic tumor cells, leading to the hypothesis that PMNs could contribute to the invasive tumor growth. To address this issue, biopsies of patients with PDAC (n = 112) were analyzed. We found that E‐cadherin expression correlated negatively with PMN infiltration, compatible with the notion that E‐cadherin is cleaved by PMN‐derived elastase, which in turn could result in the dispersal of the tumor cells, enhanced migratory capacity and thus invasive tumor growth.     

Changes in E‐ and N‐cadherin expression in developing rat adenohypophysis

Recently, we showed that hormone‐producing cells express N‐cadherin, while folliculo‐stellate cells and marginal layer cells express E‐cadherin in the adult rat anterior pituitary gland. These cells are believed to originate from a single cell population of the adenohypophyseal placode. In the present study, we immunohistochemically examined the divergence of cadherin types during pituitary histogenesis. Pituitary glands were excised from rats of embryonic day 11 (E11) through postnatal day 60 (P60) and paraffin sections were prepared. E‐ and N‐cadherins were immunostained sequentially using monoclonal and polyclonal primary antibodies and fluorescent secondary antibodies. At E11, E‐cadherin was expressed over oral epithelium, while N‐cadherin expression was limited to the primordium of Rathke's pouch. When Rathke's pouch was formed at E13, E‐ and N‐cadherin were broadly expressed in the entire cell population. N‐cadherin was expressed particularly intensely in the layer of cells that faced the lumen. From E14 through E16, the majority of cells expressed both types of cadherins; however, the cell population to become the pars tuberalis expressed N‐cadherin but not E‐cadherin. From E18 through E20, when many hormone‐producing cells appear, the number of cells that expressed N‐cadherin alone increased. However, some cell populations in the pars distalis and multilayered marginal cells still expressed both cadherins. After birth, most of the cells came to express only one of the cadherin types. These results may suggest that undetermined adenohypophyseal cells express both E‐ and N‐cadherin, but come to express either E‐ or N‐cadherin during cytogenesis. Anat Rec, 2007. © 2007 Wiley‐Liss, Inc.     

β‐Catenin and E‐cadherin expression in stage I adult‐type granulosa cell tumour of the ovary: correlation with tumour morphology and clinical outcome

Aims: To study E‐cadherin and β‐catenin expression in stage I adult‐type granulosa cell tumours (AGCTs) and correlate the findings with tumour morphology and clinical outcome. Methods and results: The study group comprised 62 FIGO stage I AGCTs, including 48 stage IA and 14 stage IC cases. Fifty patients (80.6%) had negative clinical follow‐up over periods from 3.0 to 19.2 years (median 6.4 years), and 12 patients (19.4%) developed metastases at intervals of 3.6–16.2 years (median 8.6 years). β‐Catenin and E‐cadherin were expressed in 62 (100%) and 53 (85%) primary tumours, respectively, and staining was more consistent and intense in areas showing sex cord‐like morphology. In contrast, diffuse tumour areas often showed weak or moderate staining (β‐catenin) or were negative (E‐cadherin), and there was reduced expression of both proteins in luteinized cells. Reduced β‐catenin expression in primary tumours correlated with increased risk of recurrence (P = 0.002) and a shorter time interval to recurrence, whereas there was no correlation between E‐cadherin staining and the risk of metastases. Conclusions: Localized variations in adhesion protein expression may partly explain the diverse morphological patterns exhibited by AGCT, and reduced β‐catenin staining in primary tumours may have value as an adverse prognostic factor.     

Epigenetic silencing in non‐neoplastic epithelia identifies E‐cadherin (CDH1) as a target for chemoprevention of lobular neoplasia

Invasive lobular carcinoma (ILC) of the breast is believed to develop from in situ lesions, atypical lobular hyperplasia (ALH), and lobular carcinoma in situ (LCIS). Down‐regulation of the cell–cell adhesion protein E‐cadherin is a defining feature of lobular breast cancer (LBC) and already occurs in ALH and LCIS. Apart from mutational mechanisms, epigenetic silencing of the E‐cadherin gene (CDH1) is thought to be involved in E‐cadherin down‐regulation and has been observed at a high frequency in ILC. Whether CDH1 promoter methylation is already present in in situ lesions and thus contributes to the initiation of LBC is not established. We thus examined microdissected archived tissue from 20 LBCs by methylation‐specific PCR to determine the CDH1 methylation status of lobular lesions. Nineteen of the 20 LBCs had a hypermethylated CDH1 promoter, including 13/14 ILCs and 13/13 ALHs or LCIS. Bisulphite sequencing indicated that methylation was complete within the investigated promoter fragment. Intriguingly, CDH1 methylation was likewise present in 8/8 adjacent non‐neoplastic epithelia, but not in 6/6 mammary epithelia from healthy subjects. E‐cadherin protein and mRNA were down‐regulated in in situ lesions relative to adjacent epithelia. Together, these results indicate that CDH1 promoter methylation occurs in LBC prior to E‐cadherin down‐regulation and neoplastic formation. We thus propose that epigenetic silencing represents the first of the two hits required to silence both CDH1 alleles for LBC to develop. Because promoter methylation is in principle reversible, our findings suggest that chemoprevention of LBC by epigenetic drugs should be feasible. Furthermore, the presence of CDH1 methylation in pre‐neoplastic epithelia suggests the existence of mammary regions with increased disease susceptibility, providing an explanation for the often multifocal presentation of LBC. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.