A simple novel prognostic model for early stage oral tongue cancer.

The prognostication of patient outcome is one of the greatest challenges in the management of early stage oral tongue squamous cell carcinoma (OTSCC). This study introduces a simple histopathological model for the prognostication of survival in patients with early OTSCC. A total of 311 cases (from Finland and Brazil) with clinically evaluated early stage OTSCC (cT1-T2cN0cM0) were included in this multicentre retrospective study. Tumour budding (B) and depth of invasion (D) were scored on haematoxylin-eosin-stained cancer slides. The cut-off point for tumour budding was set at 5 buds (low <5; high ≥5) and for depth of invasion at 4mm (low <4mm; high ≥4mm). The scores of B and D were combined into one model: the BD predictive model. On multivariate analysis, a high risk score (BD score 2) correlated significantly with loco-regional recurrence (P=0.033) and death due to OTSCC (P<0.001) in early stage OTSCC. The new BD model is a promising prognostic tool to identify those patients with aggressive cases of early stage OTSCC who might benefit from multimodality treatment.

miR-340 inhibits tumor cell proliferation and induces apoptosis by targeting multiple negative regulators of p27 in non-small cell lung cancer.

MicroRNAs (miRNAs) control cell cycle progression by targeting the transcripts encoding for cyclins, CDKs and CDK inhibitors, such as p27(KIP1) (p27). p27 expression is controlled by multiple transcriptional and posttranscriptional mechanisms, including translational inhibition by miR-221/222 and posttranslational regulation by the SCF(SKP2) complex. The oncosuppressor activity of miR-340 has been recently characterized in breast, colorectal and osteosarcoma tumor cells. However, the mechanisms underlying miR-340-induced cell growth arrest have not been elucidated. Here, we describe miR-340 as a novel tumor suppressor in non-small cell lung cancer (NSCLC). Starting from the observation that the growth-inhibitory and proapoptotic effects of miR-340 correlate with the accumulation of p27 in lung adenocarcinoma and glioblastoma cells, we have analyzed the functional relationship between miR-340 and p27 expression. miR-340 targets three key negative regulators of p27. The miR-340-mediated inhibition of both Pumilio family RNA-binding proteins (PUM1 and PUM2), required for the miR-221/222 interaction with the p27 3'-UTR, antagonizes the miRNA-dependent downregulation of p27. At the same time, miR-340 induces the stabilization of p27 by targeting SKP2, the key posttranslational regulator of p27. Therefore, miR-340 controls p27 at both translational and posttranslational levels. Accordingly, the inhibition of either PUM1 or SKP2 partially recapitulates the miR-340 effect on cell proliferation and apoptosis. In addition to the effect on tumor cell proliferation, miR-340 also inhibits intercellular adhesion and motility in lung cancer cells. These changes correlate with the miR-340-mediated inhibition of previously validated (MET and ROCK1) and potentially novel (RHOA and CDH1) miR-340 target transcripts. Finally, we show that in a small cohort of NSCLC patients (n=23), representative of all four stages of lung cancer, miR-340 expression inversely correlates with clinical staging, thus suggesting that miR-340 downregulation contributes to the disease progression.

Nuclear Nrf2 expression is related to a poor survival in pancreatic adenocarcinoma.

The aim of this study was to investigate the expression of Nrf2, sulfiredoxin and DJ1 in pancreatic cancer. The expression of Nrf2, sulfiredoxin and DJ1 was studied immunohistochemically in a large set of pancreatic adenocarcinomas consisting of 103 cases. Eighty six percent of the cases showed cytoplasmic Nrf2 and 24% nuclear Nrf2 positivity. Sulfiredoxin positivity was observed in 54% and DJ1 positivity in all cases. Nuclear Nrf2 positivity had an association with sulfiredoxin (p=0.019) and was associated with a poor survival (p=0.010). Stage IV tumors tended to have a more nuclear Nrf2 expression (p=0.080). DJ1 expression was more often found in well-differentiated tumors (p=0.012), and DJ1 expression was associated with better survival (p=0.020). According to the results, nuclear Nrf2 expression predicts a worse survival in pancreatic adenocarcinoma, which is in keeping with its protection of cells against oxidative or xenobiotic stress. In accordance with Nrf2's regulation of the synthesis of sulfiredoxin, there was an association between them (p=0.019). DJ1 had no association with Nrf2, and its expression predicted a better survival of patients.

Polarity of mature human odontoblasts.

Odontoblast polarization is based on histological appearance as columnar cells with asymmetric disposition of organelles and plasma membrane domains. However, little is known about the odontoblast plasma membrane organization. We investigated odontoblast membrane polarity using influenza virus hemagglutinin and vesicular stomatitis virus glycoprotein as model proteins in mature human odontoblast organ culture. We also examined the distribution patterns of aquaporin 4 and 5, which are basolateral and apical proteins in epithelial cells, respectively. Confocal microscopy immunofluorescence and electron microscopy demonstrated that the apical markers located at the surface toward pulp and basolateral markers located at the plasma membrane of odontoblast processes. Therefore, odontoblast plasma membrane polarity was different from that in epithelial cells. Also, certain lectins stained odontoblast processes while others stained the soma, reflecting the different natures of their membrane domains. Strong ZO-1 and weaker claudin expression suggest weak tight junctions in the odontoblasts. TGF-ß1 showed a tendency to reinstate the expression of selected TJ genes, indicating that TGF-ß1 may control odontoblast cell layer integrity by controlling tight junction protein expression.

Expression of claudins relates to tumour aggressivity, location and recurrence in ependymomas.

The aim of our study was to assess the nature and importance of claudin expression in grade I-III ependymomas. The expression of claudins 2-5, 7, 10, TWIST, and ZEB1 were investigated in a series of 61 ependymomas using immunohistochemistry. All the claudins were expressed in ependymomas, except for CLDN4. CLDN5 positive tumours were associated with higher grade (p=0.049), whereas CLDN10 was lower in higher grade tumours (p=0.039). CLDN5 and CLDN3 were overexpressed in ependymomas of cerebral location (p=0.036, p=0.007, respectively). CLDN5 positive tumours showed more nuclear atypia, endothelial proliferation, mitosis, and hypercellularity (p=0.007, p=0.018, p=0.041, p=0.010, respectively). CLDN5 positivity correlated to higher proliferation (p=0.015). CLDN7 was more often positive in primary tumours (p=0.041). Positive ZEB1 expression was associated with CLDN2 negativity (p=0.031). TWIST-negative tumours were more often also CLDN5 and 10 negative (p=0.013, p=0.017, respectively). CLDN5 was related to more aggressive tumours compared to CLDN2 and 10, which tended to display a better degree of differentiation and a better prognosis. CLDN2 and CLDN5 were expressed commonly in ependymomas, while the parental ependymal cells in the central nervous system were usually negative. Evidently, claudins influence growth and differentiation in ependymomas.

Tumor tissue inhibitor of metalloproteinases-1 (TIMP-1) in hormone-independent breast cancer might originate in stromal cells, and improves stratification of prognosis together with nodal status.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is shown to be a potential marker for poor prognosis in breast cancer, but the biology of TIMP-1 is only partially understood. In this study, TIMP-1 production was studied in a co-culture model of hormone-independent breast cancer cell lines and mesenchymal stem cells mimicking the stromal components of the tumor. In addition, the prognostic value of TIMP-1 was histologically evaluated in a clinical material of 168 patients with hormone-independent breast tumors. The hormone-independent breast cancer (BC) cell lines MDA-MB-231, M4A4 and NM2C5 did not produce TIMP-1 protein in measureable quantities. Six tested primary mesenchymal stem cell lines all produced TIMP-1. Co-culturing of mesenchymal stem cells and breast cancer cells resulted in positive immunocytochemical diffuse staining for TIMP-1 for both cell types. Culturing breast cancer cells with MSC-conditioned media resulted in a positive cytoplasmic immunoreactivity for TIMP-1, and TIMP-1 protein concentration in cell lysates increased 2.7-fold (range 1.1-4.7). The TIMP-1 mRNA levels remained unaffected in BC cells. This might suggest that breast cancer cells can take up TIMP-1 produced by stromal cells and are thus displaying cellular immunoreactivity. In addition, TIMP-1 was shown to improve stratification of prognosis in clinical material.

Expression of claudins 7 and 18 in pancreatic ductal adenocarcinoma: association with features of differentiation.

AIM: This study was undertaken to evaluate the expression of claudins 7 and 18 in pancreatic ductal adenocarcinoma. METHODS AND RESULTS: Material tested included 111 operated samples and 47 additional biopsy samples consisting of 26 cases of pancreatitis, 3 cases of pancreatic intraepithelial neoplasia and 18 ductal adenocarcinomas. Samples were stained with antibodies to claudins 7 and 18 and analysed for membranous and cytoplasmic expression. Membrane bound claudin 7 and 18 expression was detected in 62 of 105 (59%) and 78 of 111 (70%) cases, respectively. Membrane bound claudin 7 and 18 were associated with large or intermediate neoplastic ducts (p=0.01, p=0.002, respectively). Well differentiated pancreatic adenocarcinomas displayed more cases with membrane bound claudin 7 or 18 immunopositivity (p=0.003, p=0.03, respectively). All pancreatic intraepithelial neoplasias studied expressed membrane bound claudin 18. Membrane bound claudin 7 or 18 positivity was not associated with survival (p=0.17, p=0.98). In the biopsy cases membrane bound claudin 18 had 100% specificity and 51% sensitivity for a tumour marker. CONCLUSION: Claudin 7 and 18 expression is related to gland size of neoplastic cells and is especially found in tumours with intermediate and large ducts and well differentiated tumours. Membrane bound claudin 18, when present, is a useful marker for diagnosis of pancreatic cancer. Claudins 7 and 18 were not associated with patient survival or spread of tumours.

Claudins 1, 3M, 3S, 4, 5 and 7 in vulvar neoplasms compared with vulvar squamous cell carcinoma.

The purpose of this study was to evaluate the expression of claudins 1, 3M (membrane-bound), 3S (cytoplasmic), 4, 5 and 7 in vulvar epithelial neoplasia (VIN I-III) and to compare those with invasive vulvar squamous cell carcinoma. Paraffin tissue sections from 73 vulvar neoplasms (12 VIN I, 12 VIN II-III and 49 vulvar carcinomas) were studied by immunohistochemistry for the expression of claudins 1, 3M, 3S, 4, 5 and 7. Claudin 1 stained strongly in all groups, whereas claudin 3M, 3S and 4 immunostaining were moderate in all groups. Claudin 7 stained strongly in all groups. Claudin 3M expression was higher in VIN I compared to carcinoma, while no difference was found between VIN I and VIN II-III or between VIN II-III and carcinoma. Claudin 1 and claudin 3S expressions also showed the same decreasing tendency from VIN towards vulvar carcinoma. Claudin 5 showed only weak staining in VIN I and VIN II-III, and positive expression was also low in the carcinoma group. Expressions of claudins 1, 3M, 3S, 4 and 7 were found in VIN and vulvar carcinoma. Changes in claudin 1 and claudin 3 expression during progression from VIN to vulvar carcinoma suggests a connection with claudin expression and differentiation of vulvar squamous cells. Claudin 5 does not seem to be important in VIN or vulvar carcinoma.

Claudin-5 overexpression correlates with aggressive behavior in serous ovarian adenocarcinoma.

BACKGROUND: Claudins are essential tight junctional proteins between adjacent epithelial, mesothelial or endothelial cells, and are responsible for the permeability of the paracellular space. The expression of claudin-5 and its correlation to ovarian cancer behavior was investigasted. MATERIALS AND METHODS: A total of 85 serous ovarian cancer tissue samples were analyzed using immunohistochemical staining. RESULTS: An association between claudin-5 expression and cancer grade (p=0.016) and advanced stage (p=0.022), strongest claudin-5 expression was found in advanced stage and high-grade carcinomas. An association between claudin-5 expression and cancer-specific (p=0.032) and overall survival (p=0.026) was also found. Only 25-30% of claudin-5-positive patients, but 60% of claudin-5-negative patiens were alive at the 5-years follow-up. CONCLUSION: Increased claudin-5 expression is associated with aggressive behavior in serous ovarian adenocarcinoma.

Expression of GATA-6 transcription factor in pleural malignant mesothelioma and metastatic pulmonary adenocarcinoma.

BACKGROUND: Malignant mesothelioma (MM) is a highly aggressive tumour with poor prognosis and limited response to therapy. New markers for the prediction of prognosis in MM and in pulmonary adenocarcinoma of the pleura are valuable. GATA-6 belongs to a six member zinc finger transcription factor family named after their recognition motif W-GATA-R. AIM: To clarify the distribution and possible function of GATA-6 transcription factor in MM and in pleural metastasis of lung adenocarcinomas. METHODS: 63 pleural MM and 36 pleural metastatic pulmonary adenocarcinomas were studied for GATA-6 expression by immunohistochemistry using tissue microarrays. Expression of GATA-6 was examined in relation to thyroid transcription factor-1 expression, survival, proliferation and apoptosis. RESULTS: Nuclear immunoreactivity for GATA-6 was stronger and more frequent in MM than in metastatic pleural adenocarcinoma. Prognosis was better in patients with GATA-6 expression when compared to those with no GATA-6 expression (p = 0.002); in the subgroup analysis the difference was significant in epithelial and sarcomatous mesothelioma. GATA-6 was not associated with spontaneous proliferation or apoptosis of the tumour cells in situ. CONCLUSION: Results suggest that GATA-6 plays a role in pleural malignancies, predicting longer survival in subgroups of MM.

Low claudin expression is associated with high Gleason grade in prostate adenocarcinoma.

The expression of claudins 1, 2, 3, 4, 5 and 7 was studied in prostate adenocarcinoma and compared to that of non-neoplastic epithelium and Gleason score of the tumors. Additionally, RT-PCR was performed for claudins 2 and 5 in three cases. Strong immunoreactivity of claudins 1, 3, 4 and 7 was seen in prostate adenocarcinoma while expression of claudins 2 and 5 was weaker. In relation to non-neoplastic glands, expression of claudins 2 and 5 was diminished. There was a significant association between the Gleason score and claudin 1 and 5 expression, these claudins were more strongly expressed in tumours with a lower Gleason score. A combined lowered claudin expression was associated with a high Gleason score and a poor prognosis. According to the results, there is a strong claudin expression in prostate adenocarcinoma, especially for claudins 3 and 4. In contrast, claudin 2 was low in neoplastic cells compared to non-neoplastic epithelium, suggesting downregulation of synthesis or altered metabolism/assembly of this protein during carcinoma development. The association of lowered claudin 1 and 5 expression and lowered overall claudin expression with tumours of a higher Gleason score suggest that claudins may modulate the histologic features of these tumors and in this way influence their biological behaviour.

Peroxiredoxins and antioxidant enzymes in pilocytic astrocytomas.

OBJECTIVE: Peroxiredoxins are antioxidant enzymes (AOEs), which are redox-regulated thiol proteins with potential effects on the growth, invasion and drug resistance of neoplastic cells. In this study, their biology and clinical significance were examined in pilocytic astrocytomas (PAs). MATERIAL AND METHODS: The expression of peroxiredoxins (Prx I-VI) was investigated in 105 PAs by the means of immunohistochemistry and compared with the expression of selected other antioxidant enzymes, cell proliferation, angiogenesis, apoptosis, p53, histopathology and patient survival. RESULTS: Peroxiredoxins were strongly expressed in general suggesting that oxidative damage and consequent defense takes place during the progression of pilocytic astrocytomas. In agreement with this hypothesis, several other AOEs correlated with the degenerative features and angiogenesis possibly associated with reactive oxygen species-derived cellular damage. Moreover, the expression of the AOEs was associated with each other indicating a concurrent activation of the enzymes. With the exception of manganese superoxide dismutase (MnSOD), a strong expression of AOEs was generally associated with higher cell proliferation. Prx VI seemed to have a positive association with a longer recurrence-free interval while other AOEs had no association with patient survival. Many AOEs, such as MnSOD, induce chemo- and radioresistance and are highly elevated in aggressive malignancies. PAs lack this confounding factor, and these tumors are treated only by surgery. CONCLUSIONS: Taken together, the results of this study on pilocytic astrocytomas suggest that the levels of Prxs and other AOEs and their related thiol proteins are generally strongly expressed in these tumors. At least Prx VI can contribute to tumor behavior which can make it a potential prognostic factor.

Claudins 1, 3, 4, 5 and 7 in esophageal cancer: loss of claudin 3 and 4 expression is associated with metastatic behavior.

The aim of the study was to elucidate the significance of claudins in surgically treated esophageal carcinoma. The expression of claudins 1, 3, 4, 5 and 7 was studied by immunohistochemistry. Tumor proliferation was assessed with Ki67 immunostaining and apoptosis by the TUNEL method and immunostaining of fragmented caspase 3. Adenocarcinomas showed significantly more cases with moderate or strong claudin 3 (p<0.001) and claudin 5 positivity (p=0.031) compared to squamous cell carcinomas. Loss of claudin 3 expression was associated with the presence of distant metastases (p=0.039). Claudins 3, 4 and 7 had a significant association with either a high apoptotic index or a high number of caspase 3-positive cells, while claudin 5 was associated with increased proliferation. In esophageal carcinoma, claudin expression may vary along with the histology of the tumor. Claudin expression may also be associated with apoptosis or proliferation, suggesting that claudins may contribute to tumor behavior and growth.

Expression profiling of PC-3 cell line variants and comparison of MIC-1 transcript levels in benign and malignant prostate.

BACKGROUND: The mechanisms behind prostate cancer progression are largely unknown, but macrophage inhibitory cytokine 1 (MIC-1) has been suggested to be involved in tumour dissemination in vivo due to its reductive effect on cell adhesion. MATERIALS AND METHODS: We used two PC-3 prostate cancer epithelial cell line variants as tools to screen for gene expression differences during prostate cancer progression by cDNA microarray analysis. Selected genes were further analysed by Northern blot analysis using mRNA isolated from prostatic cell lines and tissues. MIC-1 expression was studied by in situ hybridization in archival patient specimens containing benign and malignant prostatic tissue. RESULTS: Gene expression of human collagen type VI, basement membrane heparan sulphate proteoglycan, integrin alpha 1, and fibronectin I were remarkably decreased in suspension-adapted PC-3 (saPC-3) cells, indicating a gene expression profile of reduced cell adhesion. Asparagine synthetase, serine protease 1, stanniocalcin homologue, NAD-dependent methylene tetrahydrate dehydrogenase cyclohydrolase (NMDMC), fortilin, and MIC-1 were overexpressed in saPC-3 cells. In prostate, the MIC-1 gene was mainly expressed in cancer tissue. However, MIC-1 transcripts were detected in benign tissue areas, especially in specimens containing prostate cancer with Gleason sum scores of 5-8. A significant inverse correlation (Spearman's rho correlation coefficient -0.928**) was observed between the ratio of cancerous to benign MIC-1 expression levels and Gleason scores. CONCLUSIONS: Differential expression of the MIC-1 gene occurs during prostate cancer progression. The transcript level of the MIC-1 gene in histologically benign tissue seems to approach that in paired cancer tissue concomitant with an increasing Gleason score.

MMP-2 expression associates with CA 125 and clinical course in endometrial carcinoma.

OBJECTIVE: Matrix metalloproteinases have long been associated with aggressive behavior of several malignancies, but their role in endometrial cancer has not been conclusively established. This study aimed to evaluate the roles of matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) as prognostic factors in endometrial carcinoma and their association with CA 125 and other conventional prognostic markers. METHODS: The MMP-2 and MMP-9 immunoreactive proteins were evaluated from primary tumors of endometrial carcinoma in 266 specimens by using a specific monoclonal antibody in immunohistochemical stainings. The median follow-up time was 79 months. RESULTS: Expression of the MMP-2 and MMP-9 proteins was found in 88% and 70% of the primary tumors, respectively. Positive MMP-2 immunostaining was associated with a shortened recurrence-free (P=0.04) and cancer-specific survival (P=0.05). MMP-2 negativity was linked with a favorable prognosis; only one patient developed recurrent disease and died during the follow-up. Preoperative serum levels of CA 125 were higher in the patients presenting with tumors positive for MMP-2 than in those with negative immunostaining (P=0.03). CONCLUSIONS: We suggest that MMP-2 is linked with biologically aggressive nature of this cancer type. It seems that MMP-2, but not MMP-9, has some prognostic value in endometrial carcinoma. However, the conventional prognostic markers are superior to MMP-2 in assessing aggressive behavior and cancer-specific survival in endometrial cancer.

Extracellular superoxide dismutase has a highly specific localization in idiopathic pulmonary fibrosis/usual interstitial pneumonia.

AIMS: Recent studies suggest the importance of oxidant stress in the progression of pulmonary fibrosis. The aim of this study was to investigate extracellular superoxide dismutase (ECSOD), the major antioxidant enzyme of the extracellular matrix of human lung, in biopsy-proven idiopathic pulmonary fibrosis (IPF) related to usual interstitial pneumonia (UIP). METHODS AND RESULTS: Fibrotic areas and fibroblastic foci in UIP lungs were notable for absence of ECSOD by immunohistochemistry. Western blotting showed significantly lowered immunoreactivity of ECSOD in fibrotic compared with non-fibrotic areas of the diseased lung. The only cell type that showed intense ECSOD positivity in UIP was the interstitial mast cell. In order to investigate the mechanism for ECSOD depletion in fibrotic areas, alveolar epithelial cells were exposed to tumour necrosis factor-alpha and transforming growth factor (TGF)-beta1; TGF-beta suggested a trend towards decreased synthesis. Patients with UIP were also assessed to determine whether this disease is associated with a naturally occurring mutation in ECSOD (Arg213Gly) which leads to a loss of tissue binding of ECSOD. No significant differences could be found in the allele or genotype frequencies of this polymorphism between 63 UIP patients and 61 control subjects. CONCLUSION: Overall, consistent with several other antioxidant enzymes, ECSOD is very low in fibrotic areas of UIP, which may further increase the oxidant burden in this disease.

Oxidative/nitrosative stress and peroxiredoxin 2 are associated with grade and prognosis of human renal carcinoma.

Peroxiredoxins (Prxs) 1-6 were assessed in 138 renal cell carcinomas (RCC) using immunohistochemistry and selected samples by Western blotting analysis. Oxidative/nitrosative damage was evaluated using nitrotyrosine immunoreactivity. The expressions of Prxs were correlated with tumor grade and survival and nitrotyrosine reactivity. Non-malignant kidney tubular cells showed positivity with variable intensity for all six Prxs. In RCCs, most cases were positive for Prxs 1 and 2, while only 15-20% of tumors showed expression for Prxs 3 and 4. Prx 2 was associated with tumors of a lower grade (p=0.009) and with a lower frequency of distant metastases (p=0.046). Patients with tumors expressing Prx2 had better prognosis (p=0.027). Instead, nitrotyrosine was significantly associated with high grade tumors (p=0.001). Compared with the non-malignant kidney tubular cells, low Prx expression in the tumor cells can make them more susceptible to oxidative damage. Prx 2 was more abundantly expressed in low grade tumors, suggesting that this protein could play a role in preventing the development of oxidative damage, which in turn can lead to the activation of pathways leading to aggressive tumors.

Claudins in differential diagnosis between mesothelioma and metastatic adenocarcinoma of the pleura.

AIM: To study the expression of claudins in mesothelioma and metastatic pleural adenocarcinoma. METHODS: Immunohistochemical staining of claudins 1, 2, 3, 4, 5, and 7 was studied in 35 malignant mesotheliomas and the expression compared with 24 cases of pleural metastatic adenocarcinoma. All cases were also immunostained with calretinin. RESULTS: Claudin 1, 2, 3, 4, 5, and 7 expression was seen in 40%, 80%, 18%, 23%, 14%, and 43% of mesotheliomas, respectively, while the corresponding figures for adenocarcinoma were 100%, 88%, 90%, 100%, 50%, and 92%. Claudins 1, 3, 4, 5, and 7 were significantly less positive in mesothelioma than in metastatic adenocarcinoma, while no difference was observed for claudin 2. Claudins 1, 3, 4, 5, and 7 were also inversely associated with calretinin positivity. Sarcomatoid and biphasic mesothelioma subtypes appeared more negative for these claudins than pure epithelioid subtypes. Claudin expression was not associated with survival of patients with malignant mesotheliomas. CONCLUSIONS: The results show that malignant mesotheliomas have a lower expression of claudins 1, 3, 4, 5, and 7 than adenocarcinomas, and their expression could thus be used as an adjunct in differential diagnosis between the two. The difference was most evident for claudins 3 and 4, which were nearly as good as calretinin in mesothelioma detection. Sarcomatoid and biphasic mesotheliomas showed expression of these claudins only occasionally, which could be due to or contribute to their less epithelial appearance.