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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.

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.

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.

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.

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.

Background: There are no clinically useful biomarkers predictive of brain metastases (BM) in breast cancer. In this study, we investigated the correlation between expression of selected proteins in the primary tumor and the risk of BM in patients with metastatic breast cancer (MBC)., Methods: The study included 198 MBC patients (96 with and 102 without BM). Using tissue microarrays derived from the primary tumor, we assessed by immunohistochemical expression of ER, PR, HER2, Ki-67, CK5/6, EGFR, HER3, CXCR4, Rad51, E-cadherin, and claudin 3 and 4., Results: Ki-67 ≥14% (hazard ratio [HR] 2.76; P < 0.001), cytoplasmic expression of Rad51 (HR 1.87; P = 0.014) and ER-negativity (HR 1.72; P = 0.029) were associated with increased risk of BM in the multivariate analysis. A three-biomarker profile including ER, Ki-67 and Rad51 vs. other subtypes combined yielded an HR of 4.43 (P < 0.001)., Conclusions: ER-negativity, cytoplasmic expression of Rad51 and high Ki-67 are associated with increased risk of BM., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Claudins are tight junction proteins regulating the paracellular permeability of cell layers. We investigated the expression of claudins 1, 2, 3, 4, 5 and 7 in a sample set consisting of a total of 93 cases representing normal skin, actinic keratoses and squamous cell carcinomas of the skin. There were several changes found in claudin expression. Claudin 1 appeared to be progressively decreased in solar keratosis and skin squamous cell carcinomas compared to normal skin while expression of claudin 2 was increased. With claudins 3 and 5 occasional immunoreactivity was found in squamous cell carcinomas. Claudins 4 and 7 were variably expressed in skin neoplasia compared to normal skin. According to the results expression of claudins 1 and 2 change in parallel with the severity of the epidermal preneoplastic and neoplastic lesions thus probably influencing the disturbed epithelial polarity characteristic of these lesions. Claudin 1 under- and claudin 2 overexpression also lead to a leakier epithelial barrier function of the skin with a resulting damage to skin epithelial resistance. Other claudins investigated in this study did not show progressive changes even though occasional overexpression of them was found in skin squamous cell carcinoma.

Distinguishing between peritoneal epithelioid mesotheliomas and papillary serous carcinomas involving the peritoneum can be difficult on routine histological preparations, but this differential diagnosis can be facilitated by the use of immunohistochemistry. Recent investigations have indicated that PAX8, PAX2, claudin-4, and h-caldesmon are immunohistochemical markers that can assist in distinguishing between these two malignancies; however, much of the information published on the value of these markers is either insufficient or contradictory. The purpose of this study is to resolve some of the existing controversies and to fully determine the practical value of these markers for assisting in the differential diagnosis between peritoneal mesotheliomas and serous carcinomas. In order to do so, a total of 40 peritoneal epithelioid mesotheliomas and 45 serous carcinomas (15 primary, 30 metastatic to the peritoneum) were investigated. PAX8 and PAX2 nuclear positivity was demonstrated in 42 (93%) and 25 (56%) of the serous carcinomas, respectively, whereas none of the mesotheliomas expressed either marker. Forty-four (98%) of the serous carcinomas exhibited claudin-4 reactivity along the cell membrane, whereas none of the mesotheliomas were positive for this marker. All of the serous carcinomas and mesotheliomas were negative for h-caldesmon. Based on these results, it is concluded that PAX8 and claudin-4 have a higher sensitivity and specificity for assisting in discriminating between peritoneal epithelioid mesotheliomas and serous carcinomas when compared with all of the other positive carcinoma markers that are, at present, recommended to be included in the immunohistochemical panels used in this differential diagnosis. Even though it is highly specific, PAX2 has little practical value in the diagnosis of peritoneal epithelioid mesotheliomas as its sensitivity is low. The h-caldesmon is not useful.

The control of permeability through the paracellular route has been paid great attention to for enhanced bioavailability of macromolecular and hydrophilic drugs. The paracellular permeability is controlled by tight junctions (TJ), and claudins are the major constituents of TJ. Despite numerous studies on TJ modulation, the dynamics is not well understood, although it could be crucial for clinical applications. Here, we studied the time (t) course of electrical conductivity (Σ) in a monolayer of Madin-Darby canine kidney (MDCK) and Caco-2 cells upon treatment with modulators, the C-terminus fragments of Clostridium perfringens enterotoxin (C-CPE) and sodium caprate (C10). For C-CPE treatment, Σ remains approximately constant, then starts increasing at t=tc (percolation threshold). For C10, on the other hand, Σ increases to 1.6-2.0 fold of the initial value, stays constant, and then starts increasing again for both MDCK and Caco-2 cells at t=tc. We find that this behavior can be explained within a framework of percolation, where Σ shows a logarithmic dependence on t-tc with the power of μ; μ denotes the critical exponent. We obtain μ=1.1-1.2 regardless of cell type or modulator. Notably, μ depends only on the dimensionality (d) of the system, and these values correspond to those for d=2. Percolation is thus the operative mechanism for the increase in Σ through TJ modulation. The findings provide fundamental knowledge, not only on controlled drug delivery, but also on bio-nanotechnologies including the fabrication of biological devices.