Attila Marcell Szász

Expression of tight junction protein claudin-4 in Basal-Like breast carcinomas

Claudins (CLDN) are tight junction proteins which contribute to the paracellular transport and ionic permeability of various epithelia. In recent years they came into focus for their suggested role in carcinogenesis and possible role in cancer therapy. According to our previous studies, in breast tissue CLDN4 is also related to the level of cellular differentiation. Thirty-eight estrogen (ER) and progesterone receptor (PgR) negative, HER2/neu negative, but cytokeratin 5/6 positive basal-like—mainly grade 3—breast carcinomas were compared with twenty-one grade 1, twenty-five grade 2 and twenty grade 3 non-basal-like invasive breast carcinomas, in respect to their CLDN4 expression. The immunohistochemical reactions were evaluated both semiquantitatively and by morphometrical analysis. Statistically significant difference (p = 0.001) was observable regarding CLDN4 expression in the basal-like group as compared to grade 1 and 2 cancers. Further, CLDN4 expression was significantly higher (p = 0.017) in the basal-like compared with the non-basal-like grade 3 carcinomas. Our results suggest that basal-like carcinomas are a subset of breast cancer with high level of CLDN4 protein expression. The finding is in accordance with our former observation that CLDN4 is indeed related to cellular differentiation. This observation may be seen as a further proof that basal-like carcinomas represent a separable group amongst grade 3 breast carcinomas.

Claudin-1, -2, -3, -4, -7, -8, and -10 Protein Expression in Biliary Tract Cancers

Biliary tract cancers are relatively common malignant gastrointestinal tumors in the elderly. Claudins are integral components of tight junctions that play important roles in maintaining epithelial cell polarity, controlling paracellular diffusion, and regulating cell growth and differentiation. The expression profile of claudins has been extensively characterized, but few reports exist on their expression in the normal and neoplastic biliary tract. Our aim was therefore to study claudins by IHC reactions in normal and neoplastic biliary tract samples. We detected that claudin expressions differ in the normal sample groups: the normal gallbladder strongly expressed claudin-2, −3, −4, and −10, but only weak reactions were seen in normal intrahepatic bile ducts. Although each cancer type expressed several claudins with various intensities, only claudin-4 presented especially strong immunoreactions in extrahepatic bile duct cancers and gallbladder carcinomas, whereas claudin-1 and −10 presented in intrahepatic bile duct cancers. Comparing the normal and carcinoma groups, the most significant decrease was detected in the expression of claudin-10. In conclusion, the expression pattern of claudins is different in the various parts of the normal and neoplastic biliary tract; moreover, an unequivocal decrease was detected in the carcinomas compared with their corresponding normal samples. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Identification of a claudin-4 and E-cadherin score to predict prognosis in breast cancer

The elevated expression of claudins (CLDN) and E‐cadherin (CDH‐1) was found to correlate with poor prognostic features. Our aim was to perform a comprehensive analysis to assess their potential to predict prognosis in breast cancer. The expression of CLDN‐1, ‐3–5, ‐7, ‐8, ‐10, ‐15, ‐18, and E‐cadherin at the mRNA level was evaluated in correlation with survival in datasets containing expression measurements of 1809 breast cancer patients. The breast cancer tissues of 197 patients were evaluated with tissue microarray technique and immunohistochemical method for CLDN‐1–5, ‐7, and E‐cadherin protein expression. An additional validation set of 387 patients was used to test the accuracy of the resulting prognostic score. Based on the bioinformatic screening of publicly‐available datasets, the metagene of CLDN‐3, ‐4, ‐7, and E‐cadherin was shown to have the most powerful predictive power in the survival analyses. An immunohistochemical protein profile consisting of CLDN‐2, ‐4, and E‐cadherin was able to predict outcome in the most effective manner in the training set. Combining the overlapping members of the above two methods resulted in the claudin‐4 and E‐cadherin score (CURIO), which was able to accurately predict relapse‐free survival in the validation cohort (P = 0.029). The multivariate analysis, including clinicopathological variables and the CURIO, showed that the latter kept its predictive power (P = 0.040). Furthermore, the CURIO was able to further refine prognosis, separating good versus poor prognosis subgroups in luminal A, luminal B, and triple‐negative breast cancer intrinsic subtypes. In breast cancer, the CURIO provides additional prognostic information besides the routinely utilized diagnostic approaches and factors. (Cancer Sci 2011; 102: 2248–2254)

Zonula Occludens-1, Occludin, and E-cadherin Protein Expression in Biliary Tract Cancers

The incidence of cholangiocarcinomas originating from intrahepatic and extrahepatic bile ducts, as well as of gallbladder carcinoma is increasing worldwide. The malignant transformation of biliary epithelia involves profound alterations of proteins in the intercellular junctions, among others zonula occludens-1 (ZO-1), occludin, and E-cadherin. Each plays important role in the maintenance of epithelial cell polarity and regulation of cell growth and differentiation. Our aim was to investigate ZO-1, occludin, and E-cadherin immunohistochemical reactions in tissue microarray blocks containing 57 normal and 62 neoplastic biliary tract samples. We demonstrated that the tight junction components ZO-1, occludin, and E-cadherin are downregulated in carcinomas arising from various compartments of the biliary tract (normal intrahepatic and extrahepatic bile ducts, gallbladder) as compared with their normal sites of origin. These results were confirmed by discriminant analysis yielding clear separation of the three normal sample groups from carcinomas in the corresponding locations.

Correlations of Differentially Expressed Gap Junction Connexins Cx26, Cx30, Cx32, Cx43 and Cx46 with Breast Cancer Progression and Prognosis

Background and Aims Connexins and their cell membrane channels contribute to the control of cell proliferation and compartmental functions in breast glands and their deregulation is linked to breast carcinogenesis. Our aim was to correlate connexin expression with tumor progression and prognosis in primary breast cancers. Materials and Methods Meta-analysis of connexin isotype expression data of 1809 and 1899 breast cancers from the Affymetrix and Illumina array platforms, respectively, was performed. Expressed connexins were also monitored at the protein level in tissue microarrays of 127 patients equally representing all tumor grades, using immunofluorescence and multilayer, multichannel digital microscopy. Prognostic correlations were plotted in Kaplan-Meier curves and tested using the log-rank test and cox-regression analysis in univariate and multivariate models. Results The expression of GJA1/Cx43, GJA3/Cx46 and GJB2/Cx26 and, for the first time, GJA6/Cx30 and GJB1/Cx32 was revealed both in normal human mammary glands and breast carcinomas. Within their subfamilies these connexins can form homo- and heterocellular epithelial channels. In cancer, the array datasets cross-validated each other’s prognostic results. In line with the significant correlations found at mRNA level, elevated Cx43 protein levels were linked with significantly improved breast cancer outcome, offering Cx43 protein detection as an independent prognostic marker stronger than vascular invasion or necrosis. As a contrary, elevated Cx30 mRNA and protein levels were associated with a reduced disease outcome offering Cx30 protein detection as an independent prognostic marker outperforming mitotic index and necrosis. Elevated versus low Cx43 protein levels allowed the stratification of grade 2 tumors into good and poor relapse free survival subgroups, respectively. Also, elevated versus low Cx30 levels stratified grade 3 patients into poor and good overall survival subgroups, respectively. Conclusion Differential expression of Cx43 and Cx30 may serve as potential positive and negative prognostic markers, respectively, for a clinically relevant stratification of breast cancers.

The CIN4 Chromosomal Instability qPCR Classifier Defines Tumor Aneuploidy and Stratifies Outcome in Grade 2 Breast Cancer

Purpose Quantifying chromosomal instability (CIN) has both prognostic and predictive clinical utility in breast cancer. In order to establish a robust and clinically applicable gene expression-based measure of CIN, we assessed the ability of four qPCR quantified genes selected from the 70-gene Chromosomal Instability (CIN70) expression signature to stratify outcome in patients with grade 2 breast cancer. Methods AURKA, FOXM1, TOP2A and TPX2 (CIN4), were selected from the CIN70 signature due to their high level of correlation with histological grade and mean CIN70 signature expression in silico. We assessed the ability of CIN4 to stratify outcome in an independent cohort of patients diagnosed between 1999 and 2002. 185 formalin-fixed, paraffin-embedded (FFPE) samples were included in the qPCR measurement of CIN4 expression. In parallel, ploidy status of tumors was assessed by flow cytometry. We investigated whether the categorical CIN4 score derived from the CIN4 signature was correlated with recurrence-free survival (RFS) and ploidy status in this cohort. Results We observed a significant association of tumor proliferation, defined by Ki67 and mitotic index (MI), with both CIN4 expression and aneuploidy. The CIN4 score stratified grade 2 carcinomas into good and poor prognostic cohorts (mean RFS: 83.8±4.9 and 69.4±8.2 months, respectively, p = 0.016) and its predictive power was confirmed by multivariate analysis outperforming MI and Ki67 expression. Conclusions The first clinically applicable qPCR derived measure of tumor aneuploidy from FFPE tissue, stratifies grade 2 tumors into good and poor prognosis groups.

Evaluation of microvessel density (MVD) in canine mammary tumours by quantitative immunohistochemistry of the claudin-5 molecule

In our recent investigation, angiogenesis was evaluated and quantified by immunohistochemical evaluation of microvessel density (MVD) using claudin-5 (CLDN-5) as a marker for vascular endothelium in 67 canine mammary gland tumours. Computer image analysis was used to measure the intratumoural MVD. Higher intratumoural MVD was detected in malignant simple neoplasms compared with benign tumours. Furthermore, the results of MVD were correlated with histological grade, higher grades being accompanied by higher MVD. In simple adenomas and grade I tubular-tubulopapillary simple carcinomas the intratumoural microvessels were wide and regular in shape with evident erythrocytes in their lumen. In grade III solid carcinomas the microvessels were smaller, less regular and had irregular shape, often without a distinct lumen, and isolated endothelial cells were frequently present. In the complex carcinomas MVD was low and the intratumoural microvessels were mostly irregular in shape without a distinct lumen. The evaluation of MVD by CLDN-5 immunohistochemistry may give useful additional information on the angiogenic potential of breast cancers in dogs.

Expression of Tight Junction Molecules in Breast Carcinomas Analysed by Array PCR and Immunohistochemistry

In the past few decades an enormous amount of data became known to clarify the molecular composition and architecture of tight junctions (TJs). Despite the efforts, the expression and function of several TJ genes and proteins in breast carcinoma are still not known and some of the data are contradictory. The expression of forty-four TJ associated genes was examined at mRNA level in eighteen invasive ductal breast carcinoma samples and corresponding normal breast tissues by using low density array PCR. Expressions of claudins (CLDNs) 5, 10, 16, 17, and 18, and ZO-1, ZO-2 were evaluated by immunohistochemistry as well. Using immunohistochemical phenotype as a surrogate for the genetic subtype, 11 luminal A, 3 luminal B, 3 triple negative and one HER2+ cases were included. Ten genes were significantly downregulated in tumors compared with normal breast tissues (CLDNs 5, 10, 16, 18, 19, CTNNAL1, JAM-B, ZO-1, ZO-2 and PARD3), whereas one gene (CLDN17) was significantly up-regulated in tumors when compared with normal breast. At protein level CLDNs 5, 10, 16, 18, ZO-1 and ZO-2 were downregulated in tumors as compared with normal breast tissue. CLDN17 showed variable expression in tumor tissues in comparison to normal breast. In the single HER2+ tumor when compared with the other subtypes CLDNs 5, 16, 17, 18, CTNNAL1, JAM-B, ZO-1, ZO-2 and PARD3 genes were found to be upregulated. We found altered TJ genes and proteins whose expression has not yet been associated with breast carcinoma. Our findings show a tendency of TJ genes and proteins to be downregulated in breast cancer. Further studies are necessary to examine whether the downregulation of the above mentioned TJ associated genes and proteins may contribute to the malignant progression of invasive ductal breast carcinomas.

Stromal Matrix Protein Expression Following Preoperative Systemic Therapy in Breast Cancer

Purpose: Stromal alterations are observed following preoperative systemic therapy in breast cancer. The aim of the present study was to analyze the qualitative and quantitative changes of representative tumor stroma proteins in the context of neoadjuvant therapy and the response of patients undergoing preoperative systemic therapy. Experimental Design: Fifty women receiving preoperative systemic therapy were evaluated for clinical and pathologic parameters. Clinical response was defined according to International Union against Cancer (UICC) criteria, whereas pathologic responses to preoperative systemic therapy were defined according to the Chevallier and Sataloff classifications. The expression of tenascin-C, syndecan-1, collagen IV, and smooth muscle actin proteins was investigated using morphometric analysis of immunohistochemical reactions. Quantitative reverse transcription-PCR was done to evaluate the mRNA expression level of syndecan-1 and tenascin-C. The data were compared with 20 breast cancer samples of patients not treated with preoperative systemic therapy. Results: According to UICC criteria, the expression levels of collagen IV were up-regulated in all preoperative systemic therapy–treated patients (P = 0.002). Collagen IV was up-regulated in the preoperative systemic therapy group in both Chevallier and Sataloff classifications compared with the control cases (P = 0.025 and P = 001, respectively). There were no significant differences in the expression of smooth muscle actin between the treated and nontreated groups. The syndecan-1 proteoglycan level was significantly down-regulated in the preoperative systemic therapy group (Chevallier classes P = 0.015, Sataloff classes P = 0.015). Tenascin-C was up-regulated in women with progressive disease (P = 0.005). Conclusion: We have observed that the stromal component of breast carcinomas following preoperative systemic therapy differs from the nontreated tumors, which can be evaluated with the analysis of the above mentioned proteins.

Expression and localisation of claudin-1, -2, -3, -4, -5, -7 and -10 proteins in the normal canine mammary gland.

The recently identified claudins are dominant components of tight junctions, responsible for cell adhesion, polarity and paracellular permeability. Certain claudins have been shown to have relevance in tumour development. The aim of the present study was to analyse the expression of claudin-1, -2, -3, -4, -5, -7 and -10 in normal canine mammary glands. Samples from the inguinal mammary regions of 20 non-castrated, 1-13 years old female dogs were studied. Immunohistochemical analysis was performed on conventional specimens and tissue microarrays. The results of the immunohistochemical reactions detecting claudins in tissue sections were photodocumented. The immunoreactivity of claudins was quantitatively analysed on digital images using Leica QWin morphometry software. Intense membranous immunolabelling was found for claudin-1, -3 and -7, intense membranous with non-granular cytoplasmic immunolabelling for claudin-2, moderate membranous immunolabelling for claudin-4 and -5, and weak membranous immunolabelling for claudin-10. The occurrence of tight junctions was confirmed by ultrathin section electron microscopy. The available data suggested that claudins might be proteins preserved throughout the evolution of mammals. The results of our study support the concept that they are indeed preserved, since the same type of claudins, in identical distribution, could be detected in our canine mammary tissue samples as could be found in human mammary tissue.