Membranous E-cadherin Research Articles

TWIST AND E-CADHERIN EXPRESSION IN LOWER-LIP SQUAMOUS CELL CARCINOMA

Objectives To evaluate the expression of TWIST and E-cadherin in lower-lip squamous cell carcinoma (LLSCC) and their association with clinical-pathologic parameters. Study Design A total of 59 LLSCC cases diagnosed were included in the present study. Histopathologic malignancy grading by Almagush et al. (2014) and immunohistochemical analyses followed a blind method. The collected data were submitted for statistical analysis. Results LLSCC in the early clinical stages exhibited higher median values for general (P = .035) and membrane E-cadherin expression (P = .022) compared to tumors classified in more advanced stages. Higher expression of E-cadherin (general and membrane) was observed in cases with disease-free survival after 5 years of follow-up (P < .05). Higher TWIST expression was observed in lesions classified at advanced stages displaying recurrence and a high degree of malignancy. A significant negative correlation was detected between TWIST cytoplasmic expression and E-cadherin membrane expression (P = .028). A statistically significant association between intermediate- and high-risk LLSCC cases with more advanced clinical stages (III and IV) for the histopathologic grading. Conclusions The results of the present study suggest the potential involvement of TWIST and E-cadherin in the modulation of events related to worse LLSCC prognoses. To evaluate the expression of TWIST and E-cadherin in lower-lip squamous cell carcinoma (LLSCC) and their association with clinical-pathologic parameters. A total of 59 LLSCC cases diagnosed were included in the present study. Histopathologic malignancy grading by Almagush et al. (2014) and immunohistochemical analyses followed a blind method. The collected data were submitted for statistical analysis. LLSCC in the early clinical stages exhibited higher median values for general (P = .035) and membrane E-cadherin expression (P = .022) compared to tumors classified in more advanced stages. Higher expression of E-cadherin (general and membrane) was observed in cases with disease-free survival after 5 years of follow-up (P < .05). Higher TWIST expression was observed in lesions classified at advanced stages displaying recurrence and a high degree of malignancy. A significant negative correlation was detected between TWIST cytoplasmic expression and E-cadherin membrane expression (P = .028). A statistically significant association between intermediate- and high-risk LLSCC cases with more advanced clinical stages (III and IV) for the histopathologic grading. The results of the present study suggest the potential involvement of TWIST and E-cadherin in the modulation of events related to worse LLSCC prognoses.

EFFECT OF CHOLESTEROL DEPLETION ON CADHERINS EXPRESSION IN CELL LINE OF ORAL SQUAMOUS CELL CARCINOMA

The epithelial to mesenchymal transition (EMT) is the process, in which, the neoplastic cells lose epithelial phenotype, assuming a mesenchymal aspect. Among the proteins that contribute to EMT in oral squamous cell carcinoma (OSCC) there are E-cadherin and N-cadherin. Objective: To evaluate the protein expression of E-cadherin and N-cadherin after exposure to methyl-beta-cyclodextrin (MβCD) in OSCC cell line (SCC-9). Study Design: Protein expression was determined by Western blotting and immunofluorescence. Results: The cholesterol depletion resulted in the change of cell morphology, with the acquisition of mesenchymal phenotype. The protein expression of the cadherins in the treated SCC-9 was shown to be increased in relation to the parental sample. No difference was observed in E-cadherin and N-cadherin expression (P = .543 and P = .849 respectively, Kruskal-Wallis Test). Although the negative correlation was not statistically significant (P = .898; r = -0.08, Pearson), N-cadherin showed the highest median expression (median = 2557; SD = 0.261) in relation to E-cadherin (median = 4575; SD = 1.581). Furthermore, a decreased of E-cadherin membrane expression, and nuclear and cytoplasmic N-cadherin expression gain was observed on the immunofluorescence. Conclusions: The present study demonstrated that cholesterol depletion is able to regulate the protein expression of E-cadherin and N-cadherin, favoring EMT in OSCC.

E-Cadherin Downregulation is Mediated by Promoter Methylation in Canine Prostate Cancer.

E-cadherin is a transmembrane glycoprotein responsible for cell-to-cell adhesion, and its loss has been associated with metastasis development. Although E-cadherin downregulation was previously reported in canine prostate cancer (PC), the mechanism involved in this process is unclear. It is well established that dogs, besides humans, spontaneously develop PC with high frequency; therefore, canine PC is an interesting model to study human PC. In human PC, CDH1 methylation has been associated with E-cadherin downregulation. However, no previous studies have described the methylation pattern of CDH1 promoter in canine PC. Herein, we evaluated the E-cadherin protein and gene expression in canine PC compared to normal tissues. DNA methylation pattern was investigated as a regulatory mechanism of CDH1 silencing. Our cohort is composed of 20 normal prostates, 20 proliferative inflammatory atrophy (PIA) lesions, 20 PC, and 11 metastases from 60 dogs. The E-cadherin protein expression was assessed by immunohistochemistry and western blotting and gene expression by qPCR. Bisulfite- pyrosequencing assay was performed to investigate the CDH1 promoter methylation pattern. Membranous E-cadherin expression was observed in all prostatic tissues. A higher number of E-cadherin negative cells was detected more frequently in PC compared to normal and PIA samples. High-grade PC showed a diffuse membranous positive immunostaining. Furthermore, PC patients with a higher number of E-cadherin negative cells presented shorter survival time and higher Gleason scores. Western blotting and qPCR assays confirmed the immunohistochemical results, showing lower E-cadherin protein and gene expression levels in PC compared to normal samples. We identified CDH1 promoter hypermethylation in PIA and PC samples. An in vitro assay with two canine prostate cancer cells (PC1 and PC2 cell lines) was performed to confirm the methylation as a regulatory mechanism of E-cadherin expression. PC1 cell line presented CDH1 hypermethylation and after 5-Aza-dC treatment, a decreased CDH1 methylation and increased gene expression levels were observed. Positive E-cadherin cells were massively found in metastases (mean of 90.6%). In conclusion, low levels of E-cadherin protein, gene downregulation and CDH1 hypermethylation was detected in canine PC. However, in metastatic foci occur E-cadherin re-expression confirming its relevance in these processes.

Metastatic seeding of human colon cancer cell clusters expressing the hybrid epithelial/mesenchymal state.

Emerging evidence supports the theory that tumor cell clusters efficiently metastasize to distant organs. However, the roles of epithelial-to-mesenchymal transition (EMT) in metastasizing tumor cell clusters have not yet been fully elucidated. To investigate this issue, tumor fragments were dissected from 40 colorectal cancer (CRC) patients and implanted subcutaneously into immunodeficient mice. We observed that tumors developed from the tumor fragments obtained from 28 of the 40 CRC patients. The tumors were then dissociated into cell suspensions to be orthotopically injected into secondary mice. The tumors from 13 of the 28 patients progressed. Furthermore, metastases formed spontaneously in the liver and lungs from the tumor fragments obtained from 8 of these 13 patients. Moreover, employing a mathematical analysis, we showed that tumor cell clusters seeded these metastases significantly more often than did single tumor cells. Membrane E-cadherin- and nuclear ZEB1-positive tumor cells indicating the hybrid epithelial/mesenchymal state were also detected in primary tumors of various CRC patients, and in the corresponding patient-derived xenografts (PDXs) and circulating tumor cell clusters in the bloodstreams of mice. In contrast, ZEB1 staining was barely detectable in the patient-matched liver metastases presumably developing through mesenchymal-to-epithelial transition. Inhibition of E-cadherin or ZEB1 expression by shRNA notably prevented the PDX-derived tumor organoids from colonizing the liver, when injected intrasplenically into mice, indicating E-cadherin and ZEB1 expressions to be required for their metastatic colonization. Taken together, these findings suggest that the epithelial/mesenchymal state mediates metastatic seeding of human CRC cell clusters into distant organs.

E-cadherin clone 36 nuclear staining dictates adverse disease outcome in lobular breast cancer patients

Breast cancer is a heterogeneous disease and additional biomarkers for individually predicting patient outcomes are needed. Aberrant membrane E-cadherin immunoexpression has been demonstrated in lobular breast cancer. Also, E-cadherin nuclear staining has been reported, associating with prognosis in various tumors. Here, we explore whether membrane or nuclear staining of E-cadherin has the potential to dictate prognosis of patients with lobular breast cancer. We selected a cohort of 285 consecutively diagnosed lobular breast cancer patients and performed immunohistochemistry for E-cadherin (clones 36, EP700Y, and NCH38) and P-cadherin (clone 56C1) in representative formalin-fixed paraffin-embedded blocks. All patients were female, HER2-negative and surgically treated in a single institution. Survival curves were computed by Kaplan-Meier analysis. Hazard ratios and respective 95% confidence intervals were estimated using Cox regression models. Statistical significance was set at p < 0.05. Nuclear staining for E-cadherin clone 36 was frequent (35%), contrarily to other antibodies tested. Negative correlation was found between nuclear and membrane E-cadherin clone 36 immunostaining (rs = −0.30, p < 0.001), whereas positive correlation was found between membrane immunoexpression of E-cadherin clone 36 and P-cadherin (rs = 0.31, p < 0.001). Patients with any evidence of E-cadherin clone 36 nuclear immunostaining disclosed significantly worse overall survival, disease-specific-survival and disease/progression-free survival (hazard ratio = 2.059, 95% confidence interval 1.313–3.230; hazard ratio = 1.980, 95% confidence interval 1.121–3.495; and hazard ratio = 2.341, 95% confidence interval 1.403–3.905, respectively). Differences in survival were more remarkable when considering nuclear E-cadherin immunoexpression in ≥50% tumor cells. Poorer survival was maintained in multivariable analysis, after adjusting for age, menopausal and PR status, treatment course, vascular invasion, tumor grade and stage. Our results support the use of antibodies against the cytoplasmic domain of E-cadherin, such as clone 36, which may reveal nuclear immunostaining and indicate more aggressive clinical course in patients with lobular breast cancer. We hypothesize that E-cadherin is cleaved and translocated to nucleus functioning as transcription factor.

The clinical course of actinic keratosis correlates with underlying molecular mechanisms.

Actinic keratoses (AKs) are common premalignant skin lesions triggered by excessive ultraviolet exposure. The majority of AKs regress or persist, but some progress to squamous cell carcinomas. Biomarkers associated with their persistence, progression and regression have not been characterized. We performed skin biopsies in patients with extensive actinic damage to identify biomarkers that correlate with clinical progression and regression of AKs. This was an observational study of a cohort of patients with extensive actinic damage. AKs were mapped on a clear plastic template in 26 patients at months 3, 6, 9 and 11. Biopsies were taken from randomly selected, predetermined AKs and were evaluated for p53, E-cadherin, Snail, Slug and Twist. The study is registered at Clinicaltrials.gov: NCT00027976. p53 exhibited greater expression in clinically apparent AKs (histological score 2·89 ± 1·45) than in regressed AKs (0·75 ± 0·96); P < 0·01. There was also significantly less membrane E-cadherin, the lack of which is a marker of epithelial-mesenchymal transition, in clinically apparent AKs (1·89 ± 1·81) than in sun-exposed skin (3·07 ± 1·75); P < 0·005. The E-cadherin transcription repressors Snail, Slug and Twist were increased in AKs compared with sun-exposed skin. A limitation of the study is that measurement of histological biomarkers was not a primary end point. In addition, patients were allowed to apply sunscreens. At the molecular level, loss of E-cadherin and an increase in p53 are linked to the dynamic interplay between the persistence, progression and regression of AKs. What's already known about this topic? Actinic keratoses (AKs) are common dysplastic epidermal lesions that result from chronic and excessive ultraviolet exposure. Biomarkers associated with progression and regression of AK have not been characterized. What does this study add? Decreased E-cadherin and increased p53, Snail, Slug and Twist (E-cadherin transcription factors) were associated with progression from AK to nonmelanoma skin cancer. What is the translational message? Strategies targeting these molecules may be effective in reversing rising skin cancer rates. E-cadherin, p53, Snail, Slug and Twist are potential biomarkers that may be used to assess the efficacy of existing chemopreventive agents.

Carcinoma In Situ With Plasmacytoid Features: A Clinicopathologic Study of 23 Cases.

Although there are 5 well-described morphologic patterns of (nonglandular) urothelial carcinoma in situ (CIS), we have encountered a novel pattern of flat urothelial carcinoma with plasmacytoid features characterized by a triad of morphologic findings including abnormal architecture with cellular rounding, enlarged nuclei with eccentric nuclear localization, and dense globular eosinophilic cytoplasm. A total of 23 cases of plasmacytoid CIS (mean age: 74.1 y, range: 58 to 91 y) were collected and reviewed. We excluded cases in which the diagnostic biopsy had any of the following findings admixed in the same tissue biopsy sample as the plasmacytoid CIS: traditional patterns of CIS, noninvasive glandular CIS, papillary urothelial carcinoma, or invasive carcinoma. Immunostains for CK20, CD44, p53, and e-cadherin were performed on available blocks. History of prior urothelial neoplasia, prior treatment, and clinical follow-up were obtained from medical records and pathology re-review. Immunohistochemical analysis of plasmacytoid CIS showed diffuse/strong CK20 reactivity in 96% of cases (23/24), an abnormal p53 reactivity pattern (either overexpression or "null phenotype") in 37% of cases (7/19), absence of CD44 reactivity in the neoplastic cells in 63% of cases (15/24), and retained membranous e-cadherin expression in 100% of cases (18/18). Clinical follow-up (average follow-up time: 37.7 mo, range: 7 to 115 mo) showed recurrence/new occurrence in 52% of cases (12/23), including all 4 of the 23 patients who initially presented with de novo plasmacytoid CIS (ie, no prior or concomitant urothelial neoplasia). The histologic features, the immunophenotype, the association with other forms of urothelial neoplasia, and the risk of recurrence and progression in de novo lesions support that plasmacytoid CIS represents a novel pattern of flat urothelial carcinoma. These histologic features may be more subtle than in other more typical patterns of CIS and should be carefully distinguished from therapy-related/reactive changes.

Abstract 1876: Mechanism of tyrosine kinase inhibitors resistance and role of epithelial mesenchymal transition in NSCLC

Abstract EMT is a vital process in the development of metastasis and occurs when epithelial cells lose their polarized structure, by downregulation of adherens junction proteins, E-cadherin and Claudin1, located on the cell membrane. Cells with EMT are elongated spindle-like structures due to upregulation of mesenchymal markers Vimentin and N-cadherin. EMT may be responsible for tyrosine kinase inhibitor (TKIs) resistance to epidermal growth factor receptor (EGFR) in patients with activated EGFR mutations. Our earlier studies indicate that the presence of T790M mutation may induce EMT in erlotinib resistant (ER) cells (H1975). When p120-catenin, a key EMT regulator, is no longer bound to membranous E-cadherin, a complex is formed with Kaiso factor, suppressing its transcription repressor activity and promoting oncogenesis. PRMT1, another key EMT inducer, is also overexpressed in non-small cell lung cancer (NSCLC). Cells undergoing EMT also acquire cancer stem-cell (CSC) like characteristics by expressing CSC markers ABCB1, BMI-1 and Oct-4. Thus, we investigated EMT characteristics in wild type EGFR ER H358 and H2170 cells. The modulation of EMT biomarkers was determined by immunoblotting (WB) and Quantitative real-time PCR (qPCR). Expression of CSC markers was measured using flow cytometry. PRMT1 and p120-catenin were upregulated 1.5 and 6.1-fold in H358ER cells. Hence, knockout of p120-catenin and PRMT1 were performed using CRISPR-Cas9 or an inhibitor and was examined by WB and cell proliferation assay (MTT). Other EMT regulators such as Snail, PRMT-5, Vimentin and Kaiso factor were also upregulated by 2.6 to 10.7-folds and E-cadherin and Claudin-1 were downregulated by 2.6 to 10-fold in H358ER cells. RhoA, a small GTPase downstream of p120-catenin and a negative EMT regulator, was also downregulated by 2.9-fold promoting EMT in these cells. Immunofluorescence (IF) studies showed that there was 95% colocalization of p120-catenin and Kaiso factor in H358ER cells whereas 70% colocalization was seen in H358 parental cells. In ER NSCLC cells with wild type EGFR, flow cytometry studies showed increased expression of CSC markers ABCB1, BMI-1 and Oct-4. Knockdown of PRMT1 by Furamidine increased erlotinib efficacy by 16.5%% in H358ER and 21% in H2170ER cells. Similarly, knockout of p120-catenin by CRISPR-Cas9 increased erlotinib efficacy by 19% in H358ER cells. Also, there was a 54.7% increase in erlotinib’s efficiency in p120-catenin knockout H358ER cells treated with Furamidine. Moreover, immunohistochemistry (IHC) studies with p120-catenin and PRMT-1 biomarkers showed that a higher expression of these biomarkers could be related to poor prognosis in NSCLC patients. In conclusion, EMT maybe mediated through biomarkers such as PRMT1, which also upregulates other EMT regulators, and p120-catenin, which represses E-cadherin and Kaiso factor, thus activating EMT in TKI resistant cells. Citation Format: Sanjana Singh, Nabiha H. Khan, Neelu Puri. Mechanism of tyrosine kinase inhibitors resistance and role of epithelial mesenchymal transition in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1876.

Cytoplasmic E-Cadherin Expression Is Associated With Higher Tumour Level of VEGFA, Lower Response Rate to Irinotecan-based Treatment and Poorer Prognosis in Patients With Metastatic Colorectal Cancer.

The prognostic value of vascular endothelial growth factor-A (VEGFA) and epithelial cadherin (E-cadherin) expression in patients with metastatic colorectal cancer (mCRC) is controversial. In this prospective study, patients diagnosed with mCRC between August 1, 1998, and August 30, 2003, at the Turku University Hospital, Finland were included. Expression of E-cadherin (membranous and cytoplasmic pattern) and VEGFA in tumour samples was assessed by immunohistochemistry. Tumours were classified as E-cadherin expressers if they demonstrated moderate or strong cytoplasmic or membranous staining, while those positive for VEGFA expression showed a moderate or strong cytoplasmic staining. Of particular interest was the association between membranous or cytoplasmic expression of E-cadherin and VEGFA. The value of strong VEGF-A staining and membranous or cytoplasmic expression of E-cadherin as a predictor of disease outcome over a 6-year period was another point of interest in this study. Of the 67 patients with mCRC, 43 (64%) had tumours positive for cytoplasmic E-cadherin, while in 24 cases (36%), E-cadherin expression was membranous. Strong VEGFA staining was present in half of the cases (n=36, 54% of all 67 mCRC cases). VEGFA expression was significantly correlated with cytoplasmic E-cadherin expression in that 28/36 cases of VEGFA-positive tumours were also positive for cytoplasmic E-cadherin (p=0.012). In addition, among the patients with intense VEGFA expression (n=36), those who had positive cytoplasmic E-cadherin in their tumours had a lower response-rate to first-line therapy with irinotecan, fluorouracil and leucovorin regimen: 5 out of 36 (14%) were chemosensitive. This is in contrast to the patients with VEGFA-positive tumours and membranous E-cadherin (8/36, 22% chemosensitive (p=0.004). The former group also had more ominous prognosis (p<0.001). Reduced membranous expression of E-cadherin and increased cytoplasmic E-cadherin expression predict poor survival in mCRC.

Evaluation of e-cadherin expression in gastric cancer as a prognostic marker

Introduction: Carcinoma of stomach is a common malignancy and globally stands at fourth position frequency- wise. E-cadherin, is a major cell adhesion molecule, the loss of which gives rise to invasive and metastatic properties to the cancer cells. Aim of the Study: To study the E-cadherin expression in gastric carcinomas by immuno-histochemistry and to correlate its pattern of staining with tumor differentiation, invasion, histological variants, and lymph node status. Materials and Methods: A two year prospective study was done from September 2011 to August 2013 at Kakatiya Medical College and MGM Hospital, Warangal. The resected specimens of stomach and endoscopic biopsy specimens taken from carcinoma stomach in this study period were considered. Routine histopathology and immunohistochemistry for E-cadherin were done on the sections. Results: 60 cases were studied of which 26 were gastric biopsies and 34 were gastrectomy specimens. The patient age ranged from 41-72 years and the male to female ratio was 1.6:1. There were 70% and 30% cases that showed intestinal type and diffuse type morphology respectively. Preserved E-cadherin was mainly seen in intestinal type tumors 14/42 (33.33%) with grade 3 staining, while majority of diffuse adenocarcinomas 8/18 (44.44%) were negative for E-cadherin staining. Higher stage tumors and poorly differentiated adenocarcinomas showed reduced E-cadherin staining. Cases with positive lymph nodes 22/29(75.86%) had diminished / absent E-cadherin staining. Conclusion: Normal gastric mucosa on IHC shows strong membranous E-cadherin positivity. There is a gradual decrease in intensity and percentage, and the pattern changes towards cytoplasmic staining in gastric carcinomas. Keywords: Gastric carcinoma, E –Cadherin expression, Prognostic markers, Immunohistochemistry.

Mesalamine and azathioprine modulate junctional complexes and restore epithelial barrier function in intestinal inflammation

Disruption of mucosal structure and barrier function contribute to the pathogenesis of inflammatory bowel disease (IBD). Efficacy of therapy in IBD is based on endoscopic mucosal healing, which occurs by a dynamic interplay of epithelial cell regeneration, migration and differentiation. Both mesalamine (5-ASA) and azathioprine (AZTP) promote this process through mechanisms not clearly understood. We examined molecular pathways implicated in epithelial barrier function that were altered by 5-ASA and AZTP. Paracellular permeability induced by inflammatory mediators was mitigated by both compounds through restoration of cellular anchoring complexes. 5-ASA and AZTP induced rearrangement and membranous localization of junctional proteins and modulated genes involved in tight junctions. Intestinal organoids from wildtype-mice treated with TNF-α and IL-10- deficient-mice displayed impaired epithelial barrier with loss of membranous E-cadherin and reduced Desmoglein-2 expression. These effects were counteracted by 5-ASA and AZTP. Unlike AZTP that exhibited antiproliferative effects, 5-ASA promoted wound healing in colon epithelial cells. Both affected cellular senescence, cell cycle distribution and restricted cells in G1 or S phase without inducing apoptosis. This study provides mechanistic evidence that molecular actions of 5-ASA and AZTP on intestinal epithelia are fundamental in the resolution of barrier dysfunction.

Src kinase inhibition restores E-cadherin expression in dasatinib-sensitive pancreatic cancer cells.

The Src family of non-receptor tyrosine kinases are frequently activated in pancreatic ductal adenocarcinoma (PDAC), contributing to disease progression through downregulation of E-cadherin and induction of epithelial-to-mesenchymal transition (EMT). The purpose of this study was to examine the efficacy of Src kinase inhibition in restoring E-cadherin levels in PDAC. Immunohistochemical analysis of human PDAC samples showed Src activation is inversely correlated with E-cadherin levels. Protein and mRNA levels of E-cadherin, the gene expression of its various transcriptional repressors (Zeb1, Snail, Slug, LEF-1, TWIST), and changes in sub-cellular localization of E-cadherin/β-catenin in PDAC cells were characterized in response to treatment with the Src inhibitor, dasatinib (DST). DST repressed Slug mRNA expression, promoted E-cadherin transcription, and increased total and membranous E-cadherin/β-catenin levels in drug-sensitive PDAC cells (BxPC3 and SW1990), however no change was observed in drug-resistant PANC1 cells. BxPC3, PANC1, and MiaPaCa-2 flank tumor xenografts were treated with DST to examine changes in E-cadherin levels in vivo. Although DST inhibited Src phosphorylation in all xenograft models, E-cadherin levels were only restored in BxPC3 xenograft tumors. These results suggest that Src kinase inhibition reverses EMT in drug-sensitive PDAC cells through Slug-mediated repression of E-cadherin and identifies E-cadherin as potential biomarker for determining response to DST treatment.

Immunohistochemical Studies to Examine the Diagnostic and Prognostic Implications of Epithelial-to-Mesenchymal Transition in Patients with Urothelial Carcinoma of the Bladder

In the present study, quantitative expression of epithelial-to-mesenchymal transition (EMT)–associated markers was investigated immunohistochemically and their diagnostic and prognostic significance was evaluated in patients diagnosed with non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). Immunohistochemical (IHC) staining was performed in a cohort of 65 formalin-fixed and paraffin-embedded human urinary bladder tumor tissues. Tissues’ sections were obtained from archives of the Department of Pathology at SGPGIMS, India. Epithelial marker (E-cadherin), mesenchymal markers (N-cadherin and vimentin), and EMT-activating transcription factors (ATFs) (Snail and Slug) were examined for their cellular localization (membranous/cytoplasmic/nuclear) and quantitative expressions in terms of IHC score. Expression of the aforesaid markers was statistically correlated with various clinicohistopathological variables. These variables were obtained from histopathology reports and subsequent follow-up and OPD visits of patients. Impact of these markers was assessed on recurrence-free survival (RFS) in NMIBC cases and progression-free survival (PFS) in MIBC cases. The data was analyzed using SPSS 20.0 software. Focal loss of membranous E-cadherin showed statistical relevance with hematuria and tumor grade (p < 0.001, p = 0.005: independent sample t test) in MIBC patients. Novel membranous expression of N-cadherin exhibited relevance with hematuria (p = 0.011, one sample t test) in NMIBC, while membranous expression of vimentin showed correlation with tumor grade and age (p < 0.001, p < 0.001, one sample t test) in MIBC cases. Nuclear immunopositivity of Snail showed statistical association with tumor grade in both NMIBC and MIBC cases (p < 0.001, Moses non-parametric test; p < 0.001, one sample t test) and with hematuria in MIBC cases (p = 0.007, independent sample t test). Additionally, nuclear immunopositivity of Slug showed statistical association with tumor type in NMIBC and tumor grade in MIBC cases (p < 0.001, Moses non-parametric test; p < 0.001, one sample t test). Kaplan-Meier along with logrank statistics examined association between EMT profile and RFS in NMIBC cases (p < 0.001). Significant association of EMT biomarkers with clinicohistopathological outcomes may aid urologists to correlate its dynamic functions in urothelial tumorigenesis.

High-grade Endometrial Carcinomas: Morphologic and Immunohistochemical Features, Diagnostic Challenges and Recommendations.

This review of challenging diagnostic issues concerning high-grade endometrial carcinomas is derived from the authors’ review of the literature followed by discussions at the Endometrial Cancer Workshop sponsored by the International Society of Gynecological Pathologists in 2016. Recommendations presented are evidence-based, insofar as this is possible, given that the levels of evidence are weak or moderate due to small sample sizes and nonuniform diagnostic criteria used in many studies. High-grade endometrioid carcinomas include FIGO grade 3 endometrioid carcinomas, serous carcinomas, clear cell carcinomas, undifferentiated carcinomas, and carcinosarcomas. FIGO grade 3 endometrioid carcinoma is diagnosed when an endometrioid carcinoma exhibits >50% solid architecture (excluding squamous areas), or when an architecturally FIGO grade 2 endometrioid carcinoma exhibits marked cytologic atypia, provided that a glandular variant of serous carcinoma has been excluded. The most useful immunohistochemical studies to make the distinction between these 2 histotypes are p53, p16, DNA mismatch repair proteins, PTEN, and ARID1A. Endometrial clear cell carcinomas must display prototypical architectural and cytologic features for diagnosis. Immunohistochemical stains, including, Napsin A and p504s can be used as ancillary diagnostic tools; p53 expression is aberrant in a minority of clear cell carcinomas. Of note, clear cells are found in all types of high-grade endometrial carcinomas, leading to a tendency to overdiagnose clear cell carcinoma. Undifferentiated carcinoma (which when associated with a component of low-grade endometrioid carcinoma is termed “dedifferentiated carcinoma”) is composed of sheets of monotonous, typically dyscohesive cells, which can have a rhabdoid appearance; they often exhibit limited expression of cytokeratins and epithelial membrane antigen, are usually negative for PAX8 and hormone receptors, lack membranous e-cadherin and commonly demonstrate loss of expression of DNA mismatch repair proteins and SWI-SNF chromatin remodeling proteins. Carcinosarcomas must show unequivocal morphologic evidence of malignant epithelial and mesenchymal differentiation.

Twist and E-cadherin deregulation might predict poor prognosis in lower lip squamous cell carcinoma

The aim of this study was to evaluate the expression of Twist and E-cadherin in lower lip squamous cell carcinoma (LLSCC) and their association with clinicopathologic parameters. Fifty-nine cases of LLSCC were analyzed by applying immunohistochemistry techniques in a semiquantitative manner. The systems proposed by Bryne etal., Brandwein-Gensler etal., and Almangush etal. were applied for analysis of the histopathologic malignancy grading system. Higher E-cadherin expression (general and membrane) was observed in cases presenting with disease-free survival after 5years of follow-up (P < .05). Higher Twist expression was observed in lesions classified as being in advanced stages, displaying recurrence, and having a high degree of malignancy. A significant negative correlation was detected between cytoplasmic Twist expression and membrane E-cadherin expression (P = .028). A statistically significant relationship was detected between high total Twist expression in tumors classified as high risk by Brandwein-Gensler etal., and no significant difference was observed among total, membrane, and cytoplasmic E-cadherin expressions in LLSCC cases and the 3 applied grading systems (P > .05). The results of the present study suggest the potential involvement of Twist and E-cadherin in the modulation of events related to worse prognoses in LLSCC cases.

Calcium-Sensing Receptor Regulates Epidermal Intracellular Ca2+ Signaling and Re-Epithelialization after Wounding

Extracellular Ca2+ (Ca2+o) is a crucial regulator of epidermal homeostasis and its receptor, the Ca2+-sensing receptor (CaSR), conveys the Ca2+o signals to promote keratinocyte adhesion, differentiation, and survival via activation of intracellular Ca2+ (Ca2+i) and E-cadherin-mediated signaling. Here, we took genetic loss-of-function approaches to delineate the functions of CaSR in wound re-epithelialization. Cutaneous injury triggered a robust CaSR expression and a surge of Ca2+i in epidermis. CaSR and E-cadherin were co-expressed at the cell-cell membrane between migratory keratinocytes in the nascent epithelial tongues. Blocking the expression of CaSR or E-cadherin in cultured keratinocytes markedly inhibited the wound-induced Ca2+i propagation and their ability to migrate collectively. Depleting CaSR also suppressed keratinocyte proliferation by downregulating the E-cadherin/epidermal growth factor receptor/mitogen-activated protein kinase signaling axis. Blunted epidermal Ca2+i response to wounding and retarded wound healing were observed in the keratinocyte-specific CaSR knockout (EpidCasr-/-) mice, whose shortened neo-epithelia exhibited declined E-cadherin expression and diminished keratinocyte proliferation and differentiation. Conversely, stimulating endogenous CaSR with calcimimetic NPS-R568 accelerated wound re-epithelialization through enhancing the epidermal Ca2+i signals and E-cadherin membrane expression. These findings demonstrated a critical role for the CaSR in epidermal regeneration and its therapeutic potential for improving skin wound repair.

PO-478 Prognostic significance of E-cadherin and beta-catenin expression in HPV-negative oropharyngeal squamous cell carcinomas

IntroductionMounting evidence show an increasing prevalence of human papillomavirus (HPV) in head and neck squamous cell carcinomas (HNSCC), particularly in oropharyngeal squamous cell carcinoma (OPSCC), whereas tobacco-associated tumours arising in other HNSCC subsites are decreasing. HPV infection has not only an etiological role but also a prognostic significance, since patients with HPV-positive OPSCC had a much better survival than negative cases. Although there are some important and universally accepted predictive factors in OPSCC, information on other molecular markers remains scarce and controversial. The purpose of this work was to investigate the prognostic significance of E-cadherin and β-catenin expression in surgically treated human papillomavirus HPV-negative patients with OPSCC.Material and methodsSurgical tissue specimens from 241 consecutive patients with OPSCC who underwent surgical treatment at the Hospital Universitario Central de Asturias between 1990 and 2009 were retrospectively collected. Morphologically representative areas were selected from each individual formalin-fixed paraffin-embedded tumour block for the construction of a tissue microarray (TMA), and the histological diagnosis was confirmed by an experienced pathologist. Each TMA included three cores of normal epithelium (tonsil) as an internal control. Immunohistochemical analysis of E-cadherin and β-catenin protein expression was performed on OPSCC TMAs using an automatic staining workstation Dako Autostainer with the Envision Plus system.Results and discussionsE-cadherin and β-catenin expression was evaluable in 232 cases. We found that low membranous E-cadherin, low membranous β-catenin expression, and nuclear β-catenin expression were associated with a poorer disease-specific and overall survival, although the differences were only significant for β-catenin membranous expression (p=0.024 and p=0.016, respectively). In multivariate Cox analysis, nodal metastasis and low membranous β-catenin expression were significant independent predictors of reduced disease-specific and overall survival.ConclusionTaken together, these data reveal that low membranous β-catenin expression is a significant independent predictor of both reduced disease-specific and overall survival in patients with HPV-negative OPSCC. Therefore, our findings are in line with the proposed function of E-cadherin as an invasion suppressor molecule, and as such its loss could facilitate or enhance invasion of adjacent normal tissues.

Abstract 5456: Mechanism of epithelial-mesenchymal transition in erlotinib resistance in NSCLC cells containing both wild-type and mutant EGFR

Abstract EMT is a vital process in development of metastasis and occurs when epithelial cells lose their polarized structure, by downregulation of adherens junction proteins E-cadherin, Claudin and ZO-1 on the membrane. Cells with EMT are elongated spindle like structures due to upregulation of mesenchymal markers Vimentin, N-cadherin and transcription repressor of E-cadherin, ZEB1. EMT may be responsible for Tyrosine kinase inhibitor (TKI) resistance to epidermal growth factor receptor (EGFR) in patients with activated EGFR mutations. This acquired resistance to TKIs is due to a secondary T790M mutation in the kinase domain which could be responsible for inducing EMT. An EMT regulator, p120-catenin when it is no longer bound to membranous E-cadherin forms a complex with Kaiso factor suppressing its transcription repressor activity, promoting oncogenesis. PRMT1, another EMT inducer is also overexpressed in NSCLC. Cells undergoing EMT also acquire cancer stem-cell (CSC) like characteristics by expressing a CSC marker ABCB1. Thus, we investigated EMT characteristics in wild type EGFR TKI resistant H358, H2170 cells and TKI resistant EGFR mutant H1975 cells (L858R and T790M mutation) and TKI sensitive EGFR mutant H3255 cells (L858R mutation). The modulation of EMT biomarkers was determined by immunoblotting and qPCR. Expression of ABCB1 and E-cadherin was measured using flow cytometry. Key EMT regulators such as PRMT1 and p120-catenin were upregulated 4.0 and 3.2-fold, respectively. Hence, knockdown of p120-catenin and PRMT1 were performed using siRNA or an inhibitor and examined by immunoblotting and MTT. EMT transcription factors such as Slug, Snail and Twist were also upregulated 3.18, 6.2 and 1.68-folds and E-cadherin, Claudin and ZO-1 were downregulated by 8.6, 11.6 and 15.2-folds in H1975 cells compared to H3255 cells. Also, N-cadherin, ZEB1 and Vimentin were upregulated by 4.9, 3.0 and 10.7-fold. Immunofluorescence studies with Vimentin showed that H1975 cells were elongated and have a mesenchymal phenotype compared to the H3255. 90% colocalization of p120-catenin and Kaiso factor was seen in H1975 cells whereas 10% colocalization was seen in H3255 cells. In resistant NSCLC cells with wild type and mutated EGFR, flow cytometry studies showed significant increase in expression of CSC marker ABCB1 whereas E-cadherin expression was decreased. Knockdown of PRMT1 by Furamidine, increased erlotinib efficacy by 27% in H1975 cells. Similarly, knockdown of p120-catenin by siRNA increased erlotinib efficacy by 40% in H1975 cells. In conclusion, EMT maybe mediated through biomarkers such as PRMT1, which methylate's Twist, upregulates transcription factors and p120-catenin which represses E-cadherin and Kaiso factor, activating EMT in H1975 cells. Citation Format: Sanjana Singh, Tsatsral Iderzorig, Pragya Kumar, Neelu Puri. Mechanism of epithelial-mesenchymal transition in erlotinib resistance in NSCLC cells containing both wild-type and mutant EGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5456.

Cytogenetic and immunohistochemical study of 42 pigmented microcystic chromophobe renal cell carcinoma (PMChRCC).

Pigmented microcystic chromophobe renal cell carcinoma (PMChRCC) is a recently described morphologic variant of ChRCC. We have identified 42 cases in 40 patients in the last 24years. We have investigated their clinical, morphologic, immunohistochemical, and cytogenetic features. Chromosomal abnormalities of chromosomes 7 and 17 were evaluated by automated dual-color silver-enhanced in situ hybridization on paraffin-embedded tissue. Chromosomal imbalance was defined on the basis of changes in both chromosomal index and signal distribution. The main age was 60.20years, being 34 males and 6 women. The mean tumor diameter was 4.84cm, with 39 intrarenal tumors. Grossly, the tumors were solid with a brown dark colored. Microscopically, tumors consisted of pale and eosinophilic cells arranged in microcysts or microalveolar in a cribriform pattern; there were microcalcifications and a dark brown pigment, mostly extracellular. One case showed sarcomatoid transformation. All tumors were positive for epithelial membrane antigen (EMA), Claudin 7, and E-cadherin. Monosomy of 7 and 17 chromosomes was present in 1/36 cases and 2/37 cases, respectively. Polysomy of chromosome 7 and 17 was found in 26/36 cases and in 4/37, respectively. With a median follow-up of 74.05months, 37 patients were alive without disease and two were alive with disease progression. PMChRCCs expand the morphologic spectrum of the ChRCC with an unusual immunohistochemical profile. Cytogenetically, they showed monosomy to chromosome (CHR) 17 as other ChRCCs and polysomy of CHR 7 infrequent to ChRCCs. We present the probably largest series of PMCRCC, confirming their low aggressive behavior, with exceptional sarcomatoid transformation and distant metastases.

LincRNA-RoR/miR-145 promote invasion and metastasis in triple-negative breast cancer via targeting MUC1

Triple-negative breast cancer (TNBC) was associated with high rates of cancer recurrence and metastasis and currently no available molecularly target. Accumulating evidences have established the importance of lincRNA-ROR as a marker of cancers. In order to better understand the mechanism of lincRNA-ROR in TNBC, we provided a novel molecular target into the regulatory invasion and metastasis in present research. We found that lincRNA-ROR was upregulated in TNBC cell lines and tissue samples. The aberrant expression of lincRNA-ROR was shown to increase invasion and metastasis in MDA-MB-231 and loss of function by siRNA reverse these process. Furthermore, lincRNA-ROR functions as a competing endogenous RNAs (ceRNA) which sponges miR-145 and therefore upregulate the expression of Mucin1 (MUC1). The expression of MUC1 impacted E-cadherin membrane localization. Together, MUC1 was a potential molecular target may help explain the role of lincRNA-ROR/miR-145 for invasion and metastasis in TNBC cell lines.