Prognostic impact of immunohistochemical expression of claudin-4 and epithelial mesenchymal-related markers (Snail-1) in breast invasive duct carcinoma / Impacto pronóstico de la expresión inmunohistoquímica de Claudin-4 y marcadores epiteliales-mesenquimales relacionados (Snail-1) en el carcinoma ductal infiltrante de la mama

Introduction: Although the molecular profile of the breast provides prognostic indicators, risk stratification in breast cancer continues to be a challenge. Therefore, it is mandatory to seek new prognostic markers that could aid the early diagnosis of potential metastases in biopsy samples from breast cancer; among these are increased Snail-1 and Claudin-4 expression.Objectives: The aim of this study was to analyze the correlation between Snail-1 and Claudin-4 with other clinical-pathological parameters and distinct molecular subtypes.Methods: This study included 110 patients with invasive ductal carcinoma from 2009 to January 2015. Snail-1 and Claudin-4 were assessed by immunohistochemistry in formalin-fixed paraffin-embedded tissue blocks and the data were correlated with clinical-pathological data and survival.Results: A total of 65 patients (68.2%) were positive for Snail-1 and 85 patients (77.3%) were positive for Claudin-4. High Snail-1 and high Claudin-4 were detected in high-grade tumors and were associated with lymphovascular infiltration and lymph node metastases (p<0.001 for each). There was a highly significant correlation between Snail-1, Claudin-4 expression and the molecular subtype of breast cancer (p<0.001), with higher Snail-1 expression in TNBC and Her 2/neu cases (p=0.001). Claudina-4 expression in the Her2/neu enriched subtype, Snail-1-positivity and high Claudin-4 expression were associated with recurrence (p=0.001; 0.004 respectively) among the cases studied. Snail-1 and Claudin-4 were inversely related with overall survival (p=0.001) and disease-free survival (p=0.001).Conclusion: High Snail-1 and Claudin-4 levels were associated with adverse outcomes in patients with breast cancer. (AU)

Introducción: Aunque el perfil molecular de la mama proporciona indicadores de pronóstico, la estratificación del riesgo de cáncer de mama sigue siendo un desafío y es obligatoria para buscar nuevos marcadores de pronóstico que puedan facilitar el diagnóstico temprano de metástasis potenciales en las muestras de biopsia de cáncer de mama; entre estos se encuentra la expresión creciente de Snail-1 y Claudin-4.Objetivos: El objetivo de este trabajo es estudiar la correlación de Snail-1 y Claudin-4 con otros parámetros clínico-patológicos y diferentes subtipos moleculares.Métodos: Se inscribieron 110 pacientes con carcinoma de conducto invasivo en este estudio durante el período de enero de 2009 a enero de 2015. Snail-1 y Claudin-4 fueron evaluados por inmunohistoquímica (IHC) en bloques de parafina y los datos se correlacionaron con características clínico-patológicas y de supervivencia.Resultados: Fueron positivos 75 casos (68,2%) para Snail-1 y 85 (77,3%) positivos para Claudin-4. High Snail-1 y High Claudin-4 se detectaron en tumores de alto grado y se asociaron con invasión linfovascular y metástasis en los ganglios linfáticos (p < 0,001 para cada uno). Se detectó una correlación altamente significativa entre Snail-1, la expresión de Claudin-4 y el subtipo molecular de cáncer de mama (p < 0,001), con la mayor expresión de Snail-1 en los casos de cáncer de mama triple negativo (TNBC) y Her 2/neu (p = 0,001). La expresión de Claudina-4 en subtipo Her2/neu enriched, Snail-1 positivo y alta expresión de Claudin-4 se asoció con recaída (p = 0,001; 0,004, respectivamente) entre los casos estudiados. La expresión de Snail-1 y Claudin-4 se relacionó inversamente con la SG (p = 0,001) y la SSE (p = 0,001).Conclusión: Los niveles altos de proteínas Snail-1 y Claudin-4 se asocian con resultados adversos en pacientes con cáncer de mama. (AU)

Claudin-4 shows superior specificity for mesothelioma vs non-small-cell lung carcinoma compared with MOC-31 and Ber-EP4.

The distinction of malignant mesothelioma from non-small-cell lung carcinoma (NSCLC) usually requires immunohistochemistry, but some broad-spectrum carcinoma markers stain mesotheliomas, and it remains unclear which broad-spectrum markers are most valuable for distinguishing these malignancies. Here, we directly compared the sensitivity and specificity of three broad-spectrum carcinoma markers, claudin-4, Ber-EP4, and MOC-31, for distinguishing NSCLC from mesothelioma. Immunohistochemistry was performed on tissue microarrays containing 68 epithelioid mesotheliomas, 31 sarcomatoid mesotheliomas, and 147 non-small-cell lung cancers (53 adenocarcinomas, 60 squamous cell carcinomas, 13 large-cell carcinomas, and 21 sarcomatoid carcinomas). For adenocarcinoma, squamous cell carcinoma, and large-cell carcinoma, claudin-4 staining was present in 103 of 126 cases (82%), MOC-31 staining was present in 112 of 126 cases (89%), and Ber-EP4 staining was present in 113 of 126 cases (90%); these values were not statistically different. Claudin-4 stained 0 of 68 (0%), MOC-31 stained 22 of 68 (32%), and Ber-EP4 stained 24 of 68 (35%) epithelioid mesotheliomas; thus, the specificities for NSCLC versus epithelioid mesothelioma were 100%, 68%, and 65%, respectively. Claudin-4 staining was present in 7 of 21 (33%), MOC-31 staining was present in 8 of 21 (38%), and Ber-EP4 staining was present in 5 of 21 (24%) sarcomatoid carcinomas. All three markers were negative in 12 of 21 (57%) sarcomatoid carcinomas. Sarcomatoid mesotheliomas were not stained with any of these markers. We conclude that claudin-4 has considerably greater specificity and comparable sensitivity to MOC-31 and Ber-EP4 for distinguishing NSCLC from epithelioid malignant mesothelioma. The use of all three markers may be necessary for sarcomatoid neoplasms, given their limited sensitivity.

P16 as a marker of carcinoma in effusions and peritoneal washing.

BACKGROUND: Considering the potential of p16 as a marker for diagnosis, prognosis and therapeutic response, the aim of this study was to assess its presence, via immunocytochemistry, in metastatic carcinoma of different primary sites and histological types obtained from effusions and peritoneal washings. A total of 118 samples including 85 of metastatic carcinoma and 33 samples of benign effusion/peritoneal washing were prepared by the plasma/thromboplastin method. Immunocytochemistry reactions were performed on cell block sections using antibodies against p16, claudin-4, MOC-31, calretinin, HBME and CD68. RESULTS: P16 overexpression was observed in 88.23% of all carcinoma samples. All cervix adenocarcinoma samples showed p16 overexpression. Overexpression in adenocarcinomas of ovary, lung and breast was observed in 93.75, 93.10 and 75% of the samples, respectively. Overexpression was observed in all different histological types analyzed: small cell carcinoma (lung), squamous cell carcinoma (cervical) and urothelial carcinoma (bladder). The specificity of p16 for carcinoma detection was of 96.96%. CONCLUSION: Overexpression of p16 was observed in most metastatic carcinoma, from different primary sites and histological types, obtained from effusions and peritoneal washings. Due to its high frequency of overexpression in metastatic carcinoma, p16 may play a possible role in tumor progression and it may be considered as a complementary diagnostic marker depending on histological type and primary site of carcinoma.

Immunoreactivity patterns of tight junction proteins are useful for differential diagnosis of human salivary gland tumors.

The expression pattern of tight junction proteins (TJPs) varies among organs and tumor types. In this study, we examined the immunoreactivity of claudin (CLDN)-1, -4, and -7, and JAM-A in salivary gland tumors (SGTs) by histological types and cell types to estimate their usefulness as differential diagnostic markers. Immunoreactivity of CLDN1 was higher in ductal epithelium cells of SGTs than in non-tumor tissues. Conversely, immunoreactivity of CLDN1 was significantly decreased in basal/myoepithelium cells of SGTs compared with that in non-tumor tissues. There was no significant difference between the immunoreactivity of CLDN1 in benign tumors and that in malignant tumors. Immunoreactivity of CLDN4, CLDN7, and JAM-A in ductal epithelium cells was higher in many SGTs than in non-tumor tissues. There was a difference depending on the histological type of SGT in immunoreactivity of CLDN4, CLDN7, and JAM-A in basaloid/myoepithelial cells. It was possible to classify SGTs by a hierarchical clustering using immunoreactivity of TJPs. The results suggest that an immunohistochemical marker panel including these TJPs may be useful for differential diagnosis of SGTs and that CLDN1 is associated with tumorigenesis of SGTs.

Frequent loss of claudin-4 expression in dedifferentiated and undifferentiated endometrial carcinomas.

AIMS: Dedifferentiated endometrial carcinomas (DDECs)/undifferentiated endometrial carcinomas (UECs) are aggressive endometrial cancers with frequent genomic inactivation of core components of switch/sucrose non-fermentable (SWI/SNF) complex proteins. Claudin-4, an epithelial intercellular tight junction protein, was recently found to be expressed in SWI/SNF-deficient undifferentiated carcinomas but not in SWI/SNF-deficient sarcomas. The aim of this study was to examine claudin-4 expression in UECs/DDECs and other high-grade uterine carcinomas. METHODS AND RESULTS: We examined claudin-4 expression by immunohistochemistry (clone 3E2C1) on tissue microarrays that contained 44 UECs/DDECs (24 SWI/SNF-deficient), 50 carcinosarcomas, 164 grade 3 endometrioid carcinomas, 57 serous carcinomas, and 20 clear cell carcinomas. Tumours with <5% claudin-4 expression were considered to be negative. Nearly all SWI/SNF-deficient, and most SWI/SNF-proficient, UECs/DDECs showed a complete absence of claudin-4 expression in the undifferentiated component, whereas the differentiated component in DDECs showed consistent and diffuse claudin-4 expression. Only one SWI/SNF-deficient DDEC showed focal expression of claudin-4 in the undifferentiated component, as compared with diffuse expression in the corresponding differentiated component. Claudin-4 expression was consistently absent in the sarcomatous component of carcinosarcoma, and it was absent in 24% of grade 3 endometrioid carcinomas and serous carcinomas. CONCLUSION: Claudin-4 expression can be absent or very focal in a subset of high-grade endometrial carcinomas, and is almost always absent in the undifferentiated components of SWI/SNF-deficient UECs/DDECs, despite the apparent epithelial origin in the case of DDECs. Therefore, claudin-4 expression cannot be used to infer mesenchymal or epithelial tumour origin in the endometrium. The consistent loss or down-regulation of claudin-4, a tight junction protein, in SWI/SNF-deficient UECs/DDECs further supports the undifferentiated nature of these tumours.

High-level cytoplasmic claudin 3 expression is an independent predictor of poor survival in triple-negative breast cancer.

BACKGROUND: The subtype of claudin-low breast cancer can be reliably determined only by gene-expression profiling. Attempts have been made to develop immunohistochemical surrogates, which nearly always focus on membranous claudin expression. METHODS: We assessed the immunohistochemical expression of both membranous and cytoplasmic claudins 3, 4 and 7 in a series of 197 non-metastatic breast cancers, enriched with triple-negative breast cancers (TNBCs; 60%). The expression of epithelial-to-mesenchymal transition-regulating transcription factors Sip1, Zeb1 and vimentin had previously been determined in the same material. RESULTS: In multivariate analysis, strong cytoplasmic claudin 3 expression was associated with poor relapse-free survival (RFS), disease-free survival, distant disease-free survival, breast cancer-specific survival and overall survival among TNBC patients (for RFS, RR 5.202, 95% CI 1.210-22.369, p = 0.027, vs. T-class, RR 0.663, 95% CI 0.168-2.623, p = 0.558, and N-class, RR 3.940, 95% CI 0.933-16.631, p = 0.062). Cytoplasmic claudin 3 expression was also associated with strong nuclear Sip1 expression (p = 0.000053), TNBC phenotype (p = 0.012) and within them, non-basal-like phenotype (p = 0.026). Cytoplasmic claudin 7 was associated with dismal RFS (RR 6.328, 95% CI 1.401-28.593, p = 0.016, vs. T-class, RR 0.692, 95% CI 0.242-1.982, p = 0.493, and N-class, RR 2.981, 95% CI 1.1016-8.749, p = 0.047). Low cytoplasmic expression of claudins 3, 4 and 7 together also predicted poor RFS (RR 6.070, 95% CI 1.347-27.363, p = 0.019, vs. T-class, RR 0.677, 95% CI 0.237-1.934, p = 0.467, and N-class, RR 3.167, 95% CI 1.079-9.290, p = 0.036). CONCLUSIONS: Immunohistochemical expression levels of cytoplasmic claudins 3 and 7 appear to be novel prognostic factors in TNBC.

Dedifferentiated Liposarcoma With Epithelioid/Epithelial Features.

Dedifferentiated liposarcoma (DDLPS) demonstrates a variety of growth patterns, and their histologic resemblance to other spindle cell mesenchymal tumors has been widely recognized. However, epithelioid morphology in DDLPS has only rarely been documented. Here, we report 6 cases of DDLPS with striking epithelioid/epithelial features. The patients were 5 men and 1 woman with a median age of 61 years. All tumors were located in the internal trunk. During follow-up of 1 to 41 months, local recurrence, distant metastases, and tumor-related death occurred in 4, 2, and 4 patients, respectively. Beside well-differentiated liposarcoma component and conventional high-grade spindle cell morphology, all tumors focally exhibited growth comprising small or large epithelioid cells in diffuse or sheet-like proliferation. Rhabdoid cells were present in 2 cases. All 5 tumors tested harbored MDM2 amplification. Cytokeratin and/or epithelial membrane antigen were at least focally positive in all 5 tumors tested. One case contained a small focus of novel heterologous epithelial differentiation with acinar structures, wherein cytokeratin, MOC31, and claudin-4 were diffusely expressed and H3K27me3 expression was lost. DDLPS with epithelioid/epithelial features may lead to misdiagnosis of carcinoma or mesothelioma, and their diagnosis should be based on correlation with clinicopathologic and molecular findings. The epithelioid morphology in DDLPS may suggest an aggressive behavior based on this small series. In addition, we document 2 cases of MDM2-amplified undifferentiated neoplasm with epithelioid features in the internal trunk that lacked association with well-differentiated liposarcoma histology and showed rapid clinical course. Whether these latter tumors belong to DDLPS with epithelioid features requires further study.

Expression of claudin 1, claudin 4, and claudin 7 in colorectal cancer and its relation with CLDN DNA methylation patterns.

Altered claudin expression has been described in colon, prostatic, ovarian, and breast carcinoma. However, the role of epigenetic modifications in these genes and their role in colorectal cancer is unknown. We aimed our study to investigate whether claudin protein expression and methylation of CLDN can influence the tumorigenesis of colorectal cancer. A total of 31 patients diagnosed with colorectal carcinoma was used in this study. Immunohistochemical staining was used to study protein expression in both tumor and the adjacent nonneoplastic mucosa of claudin 1, 4, and 7. To detect the DNA methylation pattern of CLDN1, 4, and 7, genomic DNA was extracted from both the tumor and the adjacent nonneoplastic mucosa. Methylation analysis was carried out using bisulfite pyrosequencing. Cell membrane staining intensity of all claudins was found significantly lower in colorectal cancer tissues when compared to paired normal mucosa (p ≤ 0.001). For claudin 4, the percentage of cells staining positively was also significantly reduced (p = 0.04). In normal mucosa, cytoplasm showed no staining for claudins in any patient, whereas in paired colorectal cancer tissues, significant cytoplasmic staining appeared both for claudin 1 (p = 0.04) and claudin 4 (p = 0.01). Tumor samples were significantly hypomethylated in CLDN1 (p < 0.05). In conclusion, our results show that CLDN1 is significantly hypomethylated in tumor samples and that the membrane staining intensity for claudin 1, 4, and 7 is significantly lower in colorectal cancer tissues than in adjacent nonneoplastic tissue. Colorectal cancer cells showed dystopic cytoplasmic location of claudins.

Immunohistochemical Antibody Panel for the Differential Diagnosis of Pancreatic Ductal Carcinoma From Gastrointestinal Contamination and Benign Pancreatic Duct Epithelium in Endoscopic Ultrasound-Guided Fine-Needle Aspiration.

OBJECTIVES: The diagnosis of pancreatic ductal adenocarcinoma (PDAC) by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) can be challenging to distinguish tumor cells from benign epithelium (BE). The aim of the present study was to set a minimal antibody panel to differentiate PDAC from contaminated BE in EUS-FNA specimens. METHODS: Immunohistochemistry using claudin 4, EZH2, Ki-67, maspin, p53, and S100P was performed on tissue microarray sections containing 53 PDACs and 33 BE as well as cell blocks of EUS-FNA including 53 PDACs and 22 BE. The positive rate was scored as 0 to 4+. The receiver operating characteristic curve was applied to determine a cutoff point, and the Classification And Regression Trees method was used to obtain a classification tree of the best panel. RESULTS: The cutoff point was 1+ for claudin 4, EZH2, Ki-67, p53, and S100P and 2+ for maspin. All BE scored 0 for p53. The classification tree revealed using p53, S100P, and claudin 4 was the most powerful. The sensitivity and specificity of the tree were 96.2% and 100% in tissue microarrays and 100% and 95.5% in EUS-FNA, respectively. CONCLUSIONS: The classification tree using p53, S100P, and claudin 4 seems to successfully distinguish PDAC from the accompanying BE.

Analysis of the expression and localization of tight junction transmembrane proteins, claudin-1, -4, -7, occludin and JAM-A, in human cervical adenocarcinoma.

OBJECTIVE: Tight junction proteins have recently been reported to be useful for distinguishing between neoplastic and non-neoplastic tissues. In this study, we evaluated the expression and localization of tight junction transmembrane proteins in human cervical adenocarcinoma and adenocarcinoma in situ (AIS), and we determined whether their expression patterns could distinguish cervical adenocarcinoma from non-neoplastic cervical glands. METHODS: Fifty-five patients with cervical adenocarcinoma or AIS were included in this study. Surgical specimens were immunohistochemically stained for claudin (CLDN) -1, -4, -7, occludin, and JAM-A. RESULTS: Significantly higher expression levels of CLDNs and JAM-A were found in cervical AIS and adenocarcinoma than in non-neoplastic glands. In cervical AIS and adenocarcinoma, localization of CLDN1 and JAM-A was extended throughout the whole cell membranes, whereas they were predominantly expressed at the most apical cell-cell junction in non-neoplastic glands. ROC curve analysis revealed that immunoreactivities of CLDN-1 or JAM-A successfully distinguished neoplasms from non-neoplastic cervical glands with high specificity (CLDN-1, 79.1%; JAM-A, 79.1%) and high sensitivity (CLDN-1, 84.1%; JAM-A, 95.5%). CONCLUSIONS: As expected, there were immunohistochemical differences between cervical adenocarcinoma and non-neoplastic cervical glands by using antibodies against tight junction transmembrane proteins. These results suggest that CLDN-1 and JAM-A are potential biomarkers for cervical adenocarcinoma.

Evidence for Bicarbonate Secretion by Ameloblasts in a Novel Cellular Model.

Formation and growth of hydroxyapatite crystals during amelogenesis generate a large number of protons that must be neutralized, presumably by HCO3 (-)ions transported from ameloblasts into the developing enamel matrix. Ameloblasts express a number of transporters and channels known to be involved in HCO3 (-)transport in other epithelia. However, to date, there is no functional evidence for HCO3 (-)transport in these cells. To address questions related to HCO3 (-)export from ameloblasts, we have developed a polarized 2-dimensional culture system for HAT-7 cells, a rat cell line of ameloblast origin. HAT-7 cells were seeded onto Transwell permeable filters. Transepithelial resistance was measured as a function of time, and the expression of transporters and tight junction proteins was investigated by conventional and quantitative reverse transcription polymerase chain reaction. Intracellular pH regulation and HCO3 (-)transport were assessed by microfluorometry. HAT-7 cells formed epithelial layers with measureable transepithelial resistance on Transwell permeable supports and expressed claudin-1, claudin-4, and claudin-8-key proteins for tight junction formation. Transport proteins previously described in maturation ameloblasts were also present in HAT-7 cells. Microfluorometry showed that the HAT-7 cells were polarized with a high apical membrane CO2 permeability and vigorous basolateral HCO3 (-)uptake, which was sensitive to Na(+)withdrawal, to the carbonic anhydrase inhibitor acetazolamide and to H2DIDS inhibition. Measurements of transepithelial HCO3 (-)transport showed a marked increase in response to Ca(2+)- and cAMP-mobilizing stimuli. Collectively, 2-dimensional HAT-7 cell cultures on permeable supports 1) form tight junctions, 2) express typical tight junction proteins and electrolyte transporters, 3) are functionally polarized, and 4) can accumulate HCO3 (-)ions from the basolateral side and secrete them at the apical membrane. These studies provide evidence for a regulated, vectorial, basolateral-to-apical bicarbonate transport in polarized HAT-7 cells. We therefore propose that the HAT-7 cell line is a useful functional model for studying electrolyte transport by ameloblasts.

Prognostic Significance of Lymphatic Vessel Density Detected by D2-40 and Its Relation to Claudin-4 Expression in Prostatic Adenocarcinoma.

Background Lymphovascular invasion is an important pathway of metastatic spread and regional lymph node metastasis is the major prognostic factor in prostatic adenocarcinoma. D2-40 is used to identify the lymphatic vessels and to assess the lymphatic vessel density (LVD). Expression of claudin-4 may be related to invasion and progression of carcinoma cells in several primary tumors. Aim To evaluate intra- and peritumoral LVD through immunohistochemical expression of D2-40 in relation to claudin-4 expression and clinicopathological parameters in prostatic adenocarcinoma. Materials and Methods Immunohistochemical staining procedure was performed on 53 paraffin-embedded blocks of radical prostatectomy specimens for prostatic adenocarcinoma using anti D2-40 and claudin-4 antibodies. Sections were evaluated for mean LVD in intratumoral and peritumoral tissues assessed by D2-40 expression. Results LVD in intratumoral tissues was significantly lower compared with peritumoral areas (P = .0001). Peritumoral mean LVD was significantly higher in cases with lymphovascular invasion (P = .041) and in cases with positive lymph node metastasis (P = .003) than intratumoral mean LVD. High claudin-4 expression was significantly correlated with high tumor grade (P = .0001), lymphovascular invasion (P = .006), and positive lymph node metastasis (P = .004). High claudin-4 expression was significantly associated with increased mean LVD in peritumoral tissues. Conclusion Increased peritumoral mean LVD in prostatic adenocarcinoma is associated with lymphovascular invasion and positive lymph node metastasis. High claudin-4 expression is associated with high tumor grade, lymphocascular invasion, positive lymph node metastasis, and high mean peritumoral LVD suggesting that D2-40 and claudin-4 may represent different mechanisms of lymphatic vessel invasion with both biomarkers is related to poor prognosis.

Distinctive immunostaining of claudin-4 in spiradenomas.

The intercellular bridges are essential structures in maintaining the histologic organization of the epithelium, while providing a very efficient way to exchange molecules between cells and transduction of the cell-to-cell and matrix-to-cell signals. Derangement in those important structures' physical integrity and/or function, which can be assessed by the presence or absence of several intercellular bridge proteins including claudin-4, E-cadherin, and ß-catenin, was found to be related to several phenomena in the path to the neoplastic transformation. However, these proteins have not been studied in the wide variety of the skin neoplasms, in detail. Herein, we immunohistochemically assessed the expression patterns of these 3 intercellular bridge proteins on a total of 86 epidermal and eccrine adnexal tumors including basal cell carcinoma, squamous cell carcinoma, poroma, spiradenoma, syringoma, and hidradenoma. We observed a selective and distinct claudin-4 expression in the ductal-type cells of all cases of spiradenomas. Similarly, in the poromas, syringomas, and hidradenomas, claudin-4 was only positive in the luminal cells of microcystic structures, although not as conspicuous as in the spiradenomas. On the other hand, E-cadherin and ß-catenin were positive in almost all types of the tumors, in a way which was not contributory to differentiate from each other. In conclusion, we think that claudin-4 can be helpful at least in making a reliable differential diagnosis of spiradenoma when overlapping morphologic features do not allow to further subclassification in the overwhelming variety of the adnexal tumors.

Claudin-4 in ovarian cancer and its relation to platinum compounds resistance / Claudina-4 en cáncer ovárico y su relación con la resistencia a compuestos de platino

Background: Claudin-4, a component of the tight junction, plays an important role in tumorigenesis and metastasis of ovarian cancer, but its role in platinum resistance has not been elucidated. Aim of the work: To determine the presence of claudin-4 in ovarian cancer tissues in relation to platinum compounds resistance. Patients and methods: Patients with advanced ovarian malignancy (FIGO stages III and IV) that have undergone primary surgery for maximal cytoreduction, followed by first line chemotherapy with platinum compounds and paclitaxel, were followed up for 6 months to determine chemotherapeutic response. Claudin-4 expression in ovarian cancer tissue resected from the patients surgically was evaluated immunohistochemically. Results: Claudin-4 is associated with more aggressive behavior of ovarian tumors and the advanced stage of the tumors. High expression of claudin-4 was found in high grade tumors, of the papillary serous subtype. High expression is linked to chemotherapeutic resistance, whereas low expression is associated with good response to first line chemotherapy. Conclusion: High claudin-4 expression can predict poor chemotherapeutic response in advanced ovarian cancer (AU)

Antecedentes: La claudina-4, componente de la zona de oclusión, desempeña un papel importante en la oncogenia y metástasis del cáncer ovárico, pero su papel en la resistencia al platino no se ha aclarado todavía. Objetivo: Establecer la presencia de claudina-4 en los tejidos de cáncer ovárico con relación a la resistencia a compuestos de platino. Pacientes y métodos: Pacientes con neoplasia maligna de ovario (clasificación FIGO fase III y IV) sometidos a una intervención citorreductora primaria seguida de quimioterapia de primera línea con compuestos de platino y paclitaxel; se realizó un seguimiento durante 6 meses para valorar la reacción a la quimioterapia. Se llevó a cabo un análisis inmunohistoquímico de la expresión de la claudina-4 en los tejidos de cáncer ovárico. Resultados: La claudina-4 se asocia con un comportamiento más agresivo de los tumores ováricos y con una fase más avanzada de los mismos. Se encontró una alta manifestación de claudina-4 en tumores de grado alto, de subtipo papiloma seroso. Se relaciona la expresión alta con la resistencia quimioterápica, mientras que una expresión baja se asocia a una buena reacción a la quimioterapia de primera línea. Conclusión: Una expresión alta de claudina-4 puede predecir una mala respuesta quimioterápica en el cáncer de ovario avanzado (AU)

Expression patterns of claudins 1, 4, and 7 and their prognostic significance in nasopharyngeal carcinoma.

PURPOSE: Tight junction (TJ) proteins in the cells organize paracellular permeability, and they have a critical role in apical cell-to-cell adhesion and epithelial polarity. In our study, the expression patterns of claudins 1, 4, and 7 and their relationship with prognosis were determined in patients with nasopharyngeal carcinoma. METHODS: Claudins 1, 4, and 7 were stained immunohistochemically in 18 biopsy samples of nasopharyngeal carcinomas that included non-neoplastic surface epithelium and dysplastic epithelium in addition to the tumor tissue. The files of these patients were scanned and the stage of disease and treatment received were obtained along with demographic data such as age and gender. RESULTS: Overexpression of claudins 1, 4, and 7 in non-neoplastic surface epithelium was found in 14 (77.7%), 16 (88.8%), and 10 (55.5%) cases respectively; in dysplastic surface epithelium overexpression was found in 8 (44.4%), 13 (72.2%), and 4 (22.2%) cases, respectively; and in invasive tumor areas overexpression was found in 13 (72.2%), 9 (50%), and 10 (55.6%) cases respectively. Increased claudin 4 expression was related to advanced stage (p=0.014). There was a significant relationship determined between claudin 4 and 7 expression and decreased survival (p=0.018, p=0.024, respectively). CONCLUSION: The fact that a statistically significant relationship was found between claudin 4 expression and advanced stage, and similarly a statistically significant relationship was found between claudin 4 & 7 expression and decreased survival gives rise to thoughts that especially claudin 4 and 7 could have different tumorigenic effects on nasopharyngeal carcinoma besides their known adhesion characteristics.

The expression patterns of tight junction protein claudin-1, -3, and -4 in human gastric neoplasms and adjacent non-neoplastic tissues.

Recently, there is growing evidence that tight junction proteins are often abnormally regulated in human tumors. The function of tight junction proteins in the maintenance of normal epithelial physiology has been well discussed, but their role in the tumorigenesis of gastric cancer is less well defined. To explore the expression distinction of the tight junction proteins claudin-1, -3, and -4 expression in the gastric cancer, the expression of claudin-1, -3, and -4 in 92 gastric cancer tissues and the non-neoplastic tissues adjacent to the tumors were examined by immunohistochemistry. Compared with adjacent non-neoplastic tissues, the expression of claudin-1 was down regulated. However, the expression of claudin-3 and claudin-4 were up-regulated in gastric cancer tissue. In addition, the expression of claudin-3 is correlated with claudin-4 expression in gastric cancer. Our present study reveals that claudin-1, -3, and -4 protein expression altered between human gastric cancers and adjacent non-neoplastic tissues.

Profile of skin barrier proteins (filaggrin, claudins 1 and 4) and Th1/Th2/Th17 cytokines in adults with atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) in adults and profile of skin barrier proteins and inflammatory cytokines. OBJECTIVE: Evaluation of the expression of skin barrier proteins such as filaggrin, claudins 1 and 4 and of circulating inflammatory cytokines (Th1/Th2/Th17) in adults with AD. METHODS: Thirty-three adult patients with AD diagnosed according to the Hanifin & Rajkacriteria, and 25 healthy controls were enrolled in the study. AD severity was measured by Eczema Area and Severity Index (EASI). Laboratory assays included immunohistochemistry analysis of skin barrier proteins, such as filaggrin, claudins 1 and 4 and interleukin-17 (IL-17) from skin samples and determination of circulating cytokine levels (IL-2, 4, 5, 6, 10, 17A, TNF and IFN-γ) by flow cytometry (Cytometric Bead Array). RESULTS: We observed a reduced expression of filaggrin and claudin 1 in lesional skin of AD patients, when compared to controls. There was an inverse correlation of filaggrin expression and disease severity. In addition, IL-17 expression was enhanced in AD patients. Similarly, higher levels of inflammatory cytokines (IL-2, 5, 6, 10, 17A and IFN-γ) were found in AD patients. CONCLUSION: Our data reinforce the role of an altered skin barrier in the pathogenesis of AD. Our results show not only reduced expression of filaggrin and claudin 1 in lesional atopic skin but also inverse correlation of filaggrin expression and disease severity. Moreover, elevation of in situ IL-17 and of circulating interleukin levels in AD emphasize the systemic, inflammatory profile of this defective skin barrier dermatosis.

Universal antibody conjugation to nanoparticles using the Fcγ receptor I (FcγRI): quantitative profiling of membrane biomarkers.

Antibodies are a class of molecules widely used in bioengineering and nanomedicine for applications involving protein recognition and targeting. Here we report an efficient method for universal conjugation of antibodies to lipid-coated nanoparticles using radially oriented FcγRIs. This method is performed in physiological solution with no additional coupling reagents, thereby avoiding problems with antibody stability and functionality. Coupling to the Fc region of the antibody avoids aggregation and polymerization allowing high yield. In addition, the antibody is oriented perpendicular to the surface so that the binding sites are fully functional. Using this method we demonstrate quantitative profiling of a panel of four membrane-bound cancer biomarkers (claudin-4, mesothelin, mucin-4, and cadherin-11) on four cell lines (Panc-1, MIA PaCa-2, Capan-1, and HPDE). We show that by designing the lipid coating to minimize aggregation and nonspecific binding, we can obtain absolute values of biomarker expression levels as number per unit area on the cell surface. This method is applicable to a wide range of technologies, including solution based protein detection assays and active targeting of cell surface membrane biomarkers.

Claudin-4 immunohistochemistry is highly effective in distinguishing adenocarcinoma from malignant mesothelioma in effusion cytology.

BACKGROUND: Adenocarcinoma can be challenging to distinguish from malignant mesothelioma in effusions, and this distinction often requires ancillary studies and clinical correlation. Immunohistochemistry for claudin-4, a tight-junction-associated protein, has recently been shown to distinguish adenocarcinoma from malignant mesothelioma, mostly in surgical specimens. Our aim was to validate and assess the immunoreactivity profile of claudin-4 in a large series of malignant effusions. METHODS: We evaluated 159 malignant effusions (84 adenocarcinomas and 75 malignant mesotheliomas). Claudin-4 immunohistochemistry was performed on cell-block paraffin sections and scored for staining intensity, staining pattern (cytoplasmic versus membranous), and percentage of positive tumor cells. Appropriate positive and negative controls were used throughout. RESULTS: All cases of mesothelioma were negative for claudin-4 (0 of 64). Eighty-three of 84 cases of adenocarcinoma were positive (99%); 1 case of serous carcinoma was negative. Most adenocarcinomas showed strong and diffuse membranous staining (71 of 84; 84%); 12 cases (14%) showed membranous staining of moderate intensity. The overall sensitivity for adenocarcinoma was 99% (83 of 84). CONCLUSIONS: Claudin-4 immunohistochemistry effectively distinguishes adenocarcinoma from malignant mesothelioma with high sensitivity and specificity in the evaluation of malignant effusions.

Expression patterns of tight junction components induced by CD24 in an oral epithelial cell-culture model correlated to affected periodontal tissues.

BACKGROUND AND OBJECTIVE: Previously we demonstrated uniformly strong expression of CD24 in the epithelial attachment to the tooth and in the migrating epithelium of the periodontitis lesion. Titers of serum antibodies autoreactive with CD24 peptide correlated with reduced severity of periodontal disease. Ligation of CD24 expressed by oral epithelial cells induced formation of tight junctions that limited paracellular diffusion. In this study, we aimed to reveal that the lack of uniform expression of tight junction components in the pocket epithelium of periodontitis lesions is likely to contribute to increased paracellular permeability to bacterial products. This is proposed as a potential driver of the immunopathology of periodontitis. MATERIAL AND METHODS: An epithelial culture model with close correspondence for expression patterns for tight junction components in periodontal epithelia was used. Immunohistochemical staining and confocal laser scanning microscopy were used to analyse patterns of expression of gingival epithelial tight junction components. RESULTS: The minimally inflamed gingival attachment was characterized by uniformly strong staining at cell contacts for the tight junction components zona occludens-1, zona occludens-2, occludin, junction adhesion molecule-A, claudin-4 and claudin-15. In contrast, the pocket epithelium of the periodontal lesion showed scattered, uneven staining for these components. This pattern correlated closely with that of unstimulated oral epithelial cells in culture. Following ligation of CD24 expressed by these cells, the pattern of tight junction component expression of the minimally inflamed gingival attachment developed rapidly. CONCLUSION: There was evidence for non-uniform and focal expression only of tight junction components in the pocket epithelium. In the cell-culture model, ligation of CD24 induced a tight junction expression profile equivalent to that observed for the minimally inflamed gingival attachment. Ligation of CD24 expressed by gingival epithelial cells by lectin-like receptors of commensal oral streptococci could mediate the phenotype of health, whereas pathogenic organisms associated with periodontal disease might not signal effectively through CD24.