HER Family Protein Expression in a Greek Population with Gastric Cancer. A Retrospective Hellenic Cooperative Oncology Group Study.

BACKGROUND: Gastric cancer is a relatively common malignancy. Recently, the presence of the human epidermal growth factor receptor 2 (HER2) was identified as a molecular target in a proportion of patients who benefited from the addition of appropriate anti-HER2 treatments. Our study explored the clinical and prognostic role of known HER family members, human epidermal growth factor receptor 1 (EGFR or HER1), HER2, HER3 and HER4. PATIENTS AND METHODS: Formalin-fixed paraffin-embedded (FFPE) tumor tissue samples from 249 gastric cancer patients were studied by immunohistochemistry for protein expression of EGFR, HER2, HER3 and HER4. RESULTS: Of the 249 evaluable patients, 32 did not have complete data of treatment details and/or follow-up and were excluded from the survival analyses. Of the 217 patients with complete treatment and follow-up data, 178 were operated and treated for early disease (group 1), while 39 for advanced disease (group 2). The frequency of positive EGFR, HER2, HER3 and HER4 protein expression in all patients was 17.5%, 11.8%, 14.8% and 32.9%, respectively. There were no differences in protein expression of any of the markers between the two groups. There were, however, statistically significant associations between HER4 and all other HER family members, as well as between HER2 and HER3 expression. Of note, EGFR-positive membranous protein expression was significantly associated with the presence of lymphovascular invasion (p=0.027) and HER3 and HER4 negative cytoplasmic protein expression with well/moderately-differentiated tumors (p=0.030 and p=0.014, respectively). None of the HER family members were of prognostic value for OS in univariate analysis. CONCLUSION: The present study confirmed the known protein expression frequencies of HER family members in gastric cancer in a Greek population. Several associations were observed among the HER family members and between clinicopathological characteristics and HER family members. Further research is needed on their exact prognostic role, as well as their therapeutic targeting.

Significance of PIK3CA Mutations in Patients with Early Breast Cancer Treated with Adjuvant Chemotherapy: A Hellenic Cooperative Oncology Group (HeCOG) Study.

BACKGROUND: The PI3K-AKT pathway is frequently activated in breast cancer. PIK3CA mutations are most frequently found in the helical (exon 9) and kinase (exon 20) domains of this protein. The aim of the present study was to examine the role of different types of PIK3CA mutations in combination with molecular biomarkers related to PI3K-AKT signaling in patients with early breast cancer. METHODS: Tumor tissue samples from 1008 early breast cancer patients treated with adjuvant chemotherapy in two similar randomized trials of HeCOG were examined. Tumors were subtyped with immunohistochemistry (IHC) and FISH for ER, PgR, Ki67, HER2 and androgen receptor (AR). PIK3CA mutations were analyzed by Sanger sequencing (exon 20) and qPCR (exon 9) (Sanger/qPCR mutations). In 610 cases, next generation sequencing (NGS) PIK3CA mutation data were also available. PIK3CA mutations and PTEN protein expression (IHC) were analyzed in luminal tumors (ER and/or PgR positive), molecular apocrine carcinomas (MAC; ER/PgR negative / AR positive) and hormone receptor (ER/PgR/AR) negative tumors. RESULTS: PIK3CA mutations were detected in 235/1008 tumors (23%) with Sanger/qPCR and in 149/610 tumors (24%) with NGS. Concordance between the two methods was good with a Kappa coefficient of 0.76 (95% CI 0.69-0.82). Lobular histology, low tumor grade and luminal A tumors were associated with helical domain mutations (PIK3CAhel), while luminal B with kinase domain mutations (PIK3CAkin). The overall incidence of PIK3CA mutations was higher in luminal as compared to MAC and hormone receptor negative tumors (p = 0.004). Disease-free and overall survival did not significantly differ with respect to PIK3CA mutation presence and type. However, a statistically significant interaction between PIK3CA mutation status and PTEN low protein expression with regard to prognosis was identified. CONCLUSIONS: The present study did not show any prognostic significance of specific PIK3CA mutations in a large group of predominantly lymph-node positive breast cancer women treated with adjuvant chemotherapy. Further analyses in larger cohorts are warranted to investigate possible differential effect of distinct PIK3CA mutations in small subgroups of patients.

Prognostic Significance of VEGFC and VEGFR1 mRNA Expression According to HER2 Status in Breast Cancer: A Study of Primary Tumors from Patients with High-risk Early Breast Cancer Participating in a Randomized Hellenic Cooperative Oncology Group Trial.

BACKGROUND: Vascular endothelial growth factor C (VEGFC) and vascular endothelial growth factor receptor 1 (VEGFR1) mRNA overexpression has recently been shown to have strong predictive and prognostic value in patients with high-risk early breast cancer undergoing adjuvant chemotherapy. The present study evaluated associations of VEGFC and VEGFR1 with human epidermal growth factor receptor 2 (HER2) and their prognostic value dependent on HER2 status. PATIENTS AND METHODS: RNA was isolated from 298 formalin-fixed paraffin-embedded tumor tissue samples from the HeCOG 10/97 (HE10/97) trial, evaluating adjuvant dose-dense sequential chemotherapy with epirubicin followed by cyclophosphamide, methotrexate and 5-fluorouracil therapy with or without paclitaxel (E-T-CMF vs. E-CMF). A fully-automated method based on magnetic beads was applied for RNA extraction, followed by one-step quantitative reverse transcription-polymerase chain reaction. RESULTS: At 13.3 years of median follow-up, 116 patients (38.9%) had experienced relapse and 115 (38.6%) had died. There were strong associations between VEGFC/VEGFR1 mRNA expression and HER2 and estrogen receptor/progesterone receptor status. In multivariate analysis, both VEGFC and VEGFR1 were found to be associated with risk for death or relapse, but such associations depended on HER2 status and treatment group. High VEGFC was a negative prognostic factor for disease-free survival [hazard ratio (HR)=1.79, 95% confidence interval (CI)=1.05-3.05, Wald's p=0.032], with a trend for overall survival (HR=1.80, 95% CI=0.94-3.47, p=0.078) in patients treated with E-CMF adjusted for clinicopathological characteristics, while high VEGFR1 was associated with increased risk for death, yet non significantly in patients with HER2-negative disease (HR=1.51, 95% CI=0.82-2.77, p=0.18), regardless of treatment. CONCLUSION: VEGFC and VEGFR1 mRNA overexpression is of prognostic value, dependent on HER2 status, in patients with high-risk early breast cancer undergoing adjuvant treatment. Among HER2-negative cases, these angiogenic markers could identify more aggressive tumors with worse prognosis. Further studies are warranted to validate VEGFC and VEGFR1 as potential biomarkers in adjuvant therapy and their use in identifying sub-groups that could benefit from anti-VEGF strategies.

Randomized phase III clinical trial comparing the combination of capecitabine and oxaliplatin (CAPOX) with the combination of 5-fluorouracil, leucovorin and oxaliplatin (modified FOLFOX6) as adjuvant therapy in patients with operated high-risk stage II or stage III colorectal cancer.

BACKGROUND: The aim of the trial was to compare two active adjuvant chemotherapy regimens in patients with early stage colorectal cancer (CRC). METHODS: Patients were assigned to oxaliplatin, leucovorin and 5-FU for 12 cycles (group A, FOLFOX6) or oxaliplatin and capecitabine for eight cycles (group B, CAPOX). Primary endpoint was disease-free survival (DFS). Tumors were classified as mismatch repair proficient (pMMR) or deficient (dMMR) according to MLH1, PMS2, MSH2 and MSH6 protein expression. KRAS exon two and BRAF V600E mutational status were also assessed. RESULTS: Between 2005 and 2008, 441 patients were enrolled, with 408 patients being eligible. After a median follow-up of 74.7 months, 3-year DFS was 79.8 % (95 % CI 76.5-83.4) in the FOLFOX group and 79.5 % (95 % CI 75.9-83.1) in the CAPOX group (p = 0.78). Three-year OS was 87.2 % (95 % CI 84.1-91.1) in the FOLFOX and 86.9 % (95 % CI 83.4-89.9) in the CAPOX group (p = 0.84). Among 306 available tumors, 11.0 % were dMMR, 34.0 % KRAS mutant and 4.9 % BRAF mutant. Multivariate analysis showed that primary site in the left colon, earlier TNM stage and the presence of anemia at diagnosis were associated with better DFS and overall survival (OS), while grade one-two tumors were associated with better OS. Finally, a statistically significant interaction was detected between the primary site and MMR status (p = 0.010), while KRAS mutated tumors were associated with shorter DFS. However, the sample was too small for safe conclusions. CONCLUSIONS: No significant differences were observed in the efficacy of FOLFOX versus CAPOX as adjuvant treatment in high-risk stage II or stage III CRC patients, but definitive conclusions cannot be drawn because of the small sample size. TRIAL REGISTRATION: ANZCTR 12610000509066 . Date of Registration: June 21, 2010.

Expression of phosphorylated Akt, mTOR and MAPK in type I endometrial carcinoma: clinical significance.

BACKGROUND/AIM: The Akt/mTOR and MAPK pathways are frequently activated in various tumor types but data on endometrial carcinoma are limited. The aim of the present study was to investigate the clinical significance of the expression of phosphorylated MAPK, Akt and mTOR (p-MAPK, p-Akt, p-mTOR) in type I endometrial carcinoma. MATERIALS AND METHODS: The study comprised of 103 formalin-fixed paraffin-embedded (FFPE) type I endometrial carcino ma cases, retrospectively retrieved and assessed by immuno histochemistry for p-MAPK, p-Akt and p-mTOR expression. The expression of these proteins was also studied in non-neoplastic endometrial tissue adjacent to the tumor. RESULTS: The expression patterns of these molecules differed between malignant and non-tumorous tissue specimens. The immuno reactivity for p-Akt was exclusively detected in the neoplastic tissues. Expression levels of p-MAPK were higher in tumors compared to non-neoplastic endometrium (p<0.001), while p-mTOR was found to be over-expressed in non-neo plastic endometrium compared to carcinomas (p=0.001). Expression of p-Akt was correlated with p-MAPK protein levels (p=0.022, r=0.229). On the other hand, no association was found with clinicopathological parameters and with disease-free (DFS) or overall survival (OS) of the patients. CONCLUSION: Our findings support the de-regulation of the PI3K/Akt/mTOR and MAPK signaling pathways in type I endometrial carcinomas suggesting involvement of these pivotal pathways in endometrial carcinogenesis.

Impact of tumor angiogenic profile on the outcome of patients with metastatic breast carcinoma treated with weekly docetaxel. A Hellenic Cooperative Oncology Group (HeCOG) study.

BACKGROUND: Metronomic taxane administration has putative antiangiogenic properties. Herein, we examined the baseline tumor angiogenic profile of patients with metastatic breast carcinoma (MBC) in a prospective-retrospective translational research study. The interplay between the angiogenic factors expressed in the tumors and their prognostic value in MBC were investigated. PATIENTS AND METHODS: Tumor tissues from patients with MBC treated with weekly docetaxel (n=159) were examined by immunohistochemistry for VEGF-A, VEGF-C, VEGFR-1, VEGFR-2, VEGFR-3 and osteopontin (OPN) and by mRNA analysis for expression of VEGF-A, VEGFxxxa, VEGFxxxb, VEGF-C, thrombospondin-1 (THBS-1), hypoxia-inducible factor-1α (HIF-1α) and von Hippel-Lindau (VHL) genes. Associations between these parameters and outcome were statistically analyzed. RESULTS: Statistically significant correlations were identified between almost all biomarkers examined in continuous form, particularly at the mRNA level: VEGF-A with VEGFxxxa (rho=0.70); VEGF-C with VEGFxxxa, VEGFxxxb and VHL (rho=0.51, 0.60 and 0.44 respectively); HIF-1α with VEGF-C and THBS1 (rho= 0.48 and 0.45). High VEGF-A mRNA was associated with worse survival (p=0.0279) and marginally with progression free survival (PFS). Intratumoral co-expression of VEGFR-1 and VEGFR-2 proteins was associated with more favorable survival (p=0.0337). In multivariate analysis, only high VEGF-A mRNA levels retained their prognostic role for worse PFS and survival (PFS: HR=2.34, 95% CI=1.25-4.40, p=0.0080; survival: HR=3.15, 95% CI=1.48-6.72, p=0.0029). CONCLUSIONS: In MBC, this study confirms the adverse prognostic effect of high intratumoral VEGF-A mRNA and reveals the combined VEGFR-1/VEGFR-2 protein expression as a potentially favorable prognosticator, which merits further evaluation in larger studies.

Expression of claudins-1, -4, -5, -7 and occludin in hepatocellular carcinoma and their relation with classic clinicopathological features and patients' survival.

BACKGROUND: Occludin and claudins are integral constituents of tight junction proteins and are de-regulated in various malignancies, including hepatocellular carcinoma (HCC). This study investigated whether expression of claudins 1, 4, 5, 7 and occludin may be used as prognostic markers for overall and disease-free survival in patients with HCC after hepatectomy. PATIENTS AND METHODS: The study included 67 hepatectomy specimens obtained from an equal number of patients with HCC who underwent partial hepatectomy at the Patras University Hospital for therapeutic reasons. Ten normal liver tissues were used as controls. Expression of claudins 1, 4, 5, 7 and occludin in liver tissues was assessed by immunochemistry. Clinicopathological features were also available for each case. RESULTS: Expression of claudins 1, 4, 5, 7 and occludin was significantly increased in HCC specimens compared to non-neoplastic liver tissues and normal controls (p<0.001 in each case) Moreover, there was a statistically significant association between low level of claudin-4 and advanced tumor grade (p=0.03). Down-regulation of claudin-1 was associated with low overall survival in univariate survival analysis (p=0.049) and Kaplan-Meier analysis (p=0.04). Multivariate analysis showed that the claudin-4 level was an independent factor for survival prognosis (p=0.01). In addition, down-regulation of claudin-4 expression was associated with increased recurrence rate and low disease-free survival rate in univariate analysis (p=0.038), Kaplan-Meier plot (p=0.013) and multivariate analysis (p=0.013). A low level of claudin-5 and high level of claudin-7 levels were independent negative prognostic factors according to multivariate analysis (p=0.015 and 0.009, respectively). CONCLUSION: The present study demonstrates that high expression of claudins 1, 4, 5 and down-regulation of claudin-7 are positive prognostic markers and are associated with good outcome and increased survival rates. Moreover, an increase in claudin-4 expression may serve as an independent positive prognostic factor for low recurrence rate after hepatectomy.

The prognostic role of preoperative serum CA 125 levels in patients with endometrial carcinoma.

PURPOSE: Previous studies have shown that elevated preoperative serum CA 125 levels strongly correlate with various clinical and pathological variables and prognosis of patients with endometrial carcinoma (EC). The aim of the present study was to evaluate the clinical significance of preoperative serum CA 125 levels in patients with EC. METHODS: A retrospective study of all EC patients treated at our institution between 1995 and 2010 with available follow-up was conducted. The preoperative serum level of CA 125 was measured in 99 patients and evaluated in relation to various clinical and pathological variables and outcome. We used the cut-off level of 20 U/ml for CA 125 on chi-square test for categorical variables. Survival analysis was performed with the use of Kaplan Meier method, the log rank test and Cox proportional hazards regression analysis. RESULTS: In the early stages of disease the mean values of CA 125 were 35 U/ml (SD±70) for stages IA-IB and 21 U/ml (SD±29) for stage IC (Mann-Whitney test for continuous variables). In advanced stages of disease (III-IV), the values of preoperative serum CA 125 levels were statistically increased, with mean value 54 U/ml (SD±44), in comparison to stages IA-IB (p=0.02) and IC (p=0.007). According to the multivariate analysis, elevated preoperative serum CA 125 level (p=0.043) and histological tumor type (p=0.004) were independent prognostic factors for disease free survival (DFS) and overall survival (OS) of patients with EC. CONCLUSION: The current study suggests that measurement of preoperative serum CA 125 is a useful clinical tool in the prognosis of patients with EC.

Targeted pathways in breast cancer: molecular and protein markers guiding therapeutic decisions.

Breast carcinoma is currently considered as a group of diseases, differing not only in histopathologic phenotype, as indicated by histologic type and grade, but also in their protein, genetic and epigenetic molecular profile. The standard of care indicates that the core information for patient management includes data on Estrogen Receptor (ER), Progesterone Receptor (PgR) and Human Epidermal Growth Factor Receptor 2 (HER2), while there is an emerging role for the proliferation marker Ki67. These indices can be provided even in low resource settings and are indispensable for prognostication and therapeutic patient management. With the progress in molecular and translational research, there is a growing body of information on the molecular subtypes of breast carcinoma and their significance, and multigene signature assays are used to dictate prognosis and guide therapeutics in high resource settings. In addition, several cellular pathways involved in tumor growth and spread are dissected and targeted in clinical trials. Among these are the p53, RB, PI3K/Akt/mTOR and Ras/MAPK pathways, alterations associated with genetic instability and epigenetic alterations including histone methylation and acetylation, DNA methylation and microRNAs expression. The tumor immune microenvironment, including the tumor infiltrating lymphocytes (TILs) is attracting significant research interest. This review summarizes the mechanisms of function of the above factors in breast tumorigenesis with emphasis on their prognostic and predictive value and their use as therapeutic targets.

Prognostic significance of ESR1 gene amplification, mRNA/protein expression and functional profiles in high-risk early breast cancer: a translational study of the Hellenic Cooperative Oncology Group (HeCOG).

BACKGROUND: Discrepant data have been published on the incidence and prognostic significance of ESR1 gene amplification in early breast cancer. PATIENTS AND METHODS: Formalin-fixed paraffin-embedded tumor blocks were collected from women with early breast cancer participating in two HeCOG adjuvant trials. Messenger RNA was studied by quantitative PCR, ER protein expression was centrally assessed using immunohistochemistry (IHC) and ESR1 gene copy number by dual fluorescent in situ hybridization probes. RESULTS: In a total of 1010 women with resected node-positive early breast adenocarcinoma, the tumoral ESR1/CEP6 gene ratio was suggestive of deletion in 159 (15.7%), gene gain in 551 (54.6%) and amplification in 42 cases (4.2%), with only 30 tumors (3%) harboring five or more ESR1 copies. Gene copy number ratio showed a significant, though weak correlation to mRNA and protein expression (Spearman's Rho <0.23, p = 0.01). ESR1 clusters were observed in 9.5% (57 gain, 38 amplification) of cases. In contrast to mRNA and protein expression, which were favorable prognosticators, gene copy number changes did not obtain prognostic significance. When ESR1/CEP6 gene ratio was combined with function (as defined by ER protein and mRNA expression) in a molecular classifier, the Gene Functional profile, it was functional status that impacted on prognosis. In univariate analysis, patients with functional tumors (positive ER protein expression and gene ratio normal or gain/amplification) fared better than those with non-functional tumors with ESR1 gain (HR for relapse or death 0.49-0.64, p = 0.003). Significant interactions were observed between gene gain/amplification and paclitaxel therapy (trend for DFS benefit from paclitaxel only in patients with ESR1 gain/amplification, p = 0.066) and Gene Functional profile with HER2 amplification (Gene Functional profile prognostic only in HER2-normal cases, p = 0.029). CONCLUSIONS: ESR1 gene deletion and amplification do not constitute per se prognostic markers, instead they can be classified to distinct prognostic groups according to their protein-mediated functional status.

Nrf2 is commonly activated in papillary thyroid carcinoma, and it controls antioxidant transcriptional responses and viability of cancer cells.

CONTEXT: The antioxidant transcription factor NFE2-related factor 2 (Nrf2), encoded by NFE2L2, has been implicated as mediator of thyroid cancer cell line resistance to proteasome inhibitors. However, the activity status of the Nrf2 pathway in human thyroid cancer remains unknown. OBJECTIVE: The aims of this study were assessment of the activity status of the Nrf2 pathway in papillary thyroid carcinoma (PTC) and investigation of its role(s) in antioxidant transcriptional responses and viability of cancer cells. DESIGN AND SETTING: We conducted retrospective immunohistochemical analyses of PTC specimens, adjacent normal tissue, and benign lesions; assays of viability and gene expression in the PTC cell lines K1 and TPC-1 after genetic/pharmacological manipulation of Nrf2; and DNA sequencing at an academic medical center. PATIENTS: The study included 42 PTC and 42 benign lesions (24 adenomas and 18 nodular hyperplasias). MAIN OUTCOME MEASURES: We assessed the abundance of Nrf2, Nqo1, Keap1, and 4HNE; cell line viability and mRNA expression of Nrf2, Nqo1, and Trdx1; and the sequence of NFE2L2, KEAP1, and BRAF. RESULTS: Nrf2 and its target Nqo1 were undetectable in normal tissue; their levels were significantly higher in PTC than in benign lesions (P < .0001 and P = .024, respectively). The Nrf2 inhibitor Keap1 was variably abundant in PTC, and its levels did not correlate with Nrf2 (P = .37), arguing against decreased levels as the mechanism for Nrf2 activation. The oxidized lipid 4HNE was more abundant in PTC than normal tissue (P < .001), indicating oxidative stress. Nrf2 mediated transcriptional antioxidant responses in both the PTC cell lines K1 and TPC-1 and in the nontransformed cell line TAD2, but it conferred a viability advantage specifically in the PTC cell lines. CONCLUSIONS: The high activity of Nrf2 in PTC warrants further exploration of this pathway's potential diagnostic, prognostic, and/or therapeutic utility in PTC.

Intestinal mucosal proliferation, apoptosis and oxidative stress in patients with liver cirrhosis.

BACKGROUND: Intestinal mucosal barrier dysfunction in liver cirrhosis and its implicated mechanisms is of great clinical importance because it is associated with the development of serious complications from diverse organs through promotion of systemic endotoxemia. AIM: The present study was designed to investigate whether enterocytes' proliferation, apoptosis and intestinal oxidative stress are altered in the intestinal mucosa of patients with compensated and decompensated liver cirrhosis. MATERIAL AND METHODS: Twelve healthy controls (group A) and twenty four cirrhotic patients at a compensated (n = 12, group B) or decompensated condition (n = 12, group C) were subjected to duodenal biopsy. In intestinal specimens mucosal apoptotic and mitotic activity and their ratio were recorded by means of morphological assessment and mucosal lipid hydroperoxides were measured. Plasma endotoxin concentration, an index of gut barrier function, was also determined. RESULTS: Cirrhotic patients presented significantly higher serum endotoxin concentrations as compared to healthy controls (P < 0.001), whilst endotoxemia was higher in decompensated disease (P < 0.05 vs. compensated cirrhosis). Intestinal mucosal mitotic count was significantly lower in patients with compensated and decompensated cirrhosis compared to controls (P < 0.01, respectively), whilst a trend towards increased apoptosis was recorded. The mitotic/apoptotic ratio was significantly reduced in groups B (P < 0.05) and C (P < 0.01) as compared to controls. Intestinal lipid peroxidation was significantly increased in decompensated cirrhotics (P < 0.001 vs. groups A and B). CONCLUSIONS: The present study demonstrates for the first time that human liver cirrhosis is associated with decreased intestinal mucosal proliferation and proliferation/apoptosis ratio even at early stages of cirrhosis and increased intestinal oxidative stress in advanced liver disease.

Altered intestinal tight junctions' expression in patients with liver cirrhosis: a pathogenetic mechanism of intestinal hyperpermeability.

BACKGROUND: Increased intestinal permeability in cirrhosis exerts a pivotal role in the pathogenesis of spontaneous bacterial peritonitis and other complications of cirrhosis through promotion of systemic endotoxemia. This study was designed to investigate whether the expression of tight junction (TJ) proteins, which regulate gut paracellular permeability, is altered in the intestinal mucosa of patients with liver cirrhosis and study its potential association with the stage of liver disease and the development of systemic endotoxemia. DESIGN: Twenty-four patients with cirrhosis at a decompensated (n = 12, group A) or compensated condition (n = 12, group B) and 12 healthy controls (group C) were subjected to duodenal biopsy. The expression of the TJ proteins occludin and claudin-1 in the intestinal epithelium was evaluated by immunohistochemistry. Plasma endotoxin concentrations were also determined. RESULTS: Patients with cirrhosis presented significantly higher serum endotoxin concentrations as compared to healthy controls (P < 0·001), whilst endotoxemia was higher in decompensated disease (P < 0·05 vs. compensated cirrhosis). Patients with decompensated and compensated cirrhosis presented significantly reduced expression of occludin and claudin-1 as compared to controls (P < 0·01, respectively). These alterations were significantly more pronounced in decompensated patients as compared to compensated (P < 0·05). Regarding occludin, in patients with cirrhosis, a specific pattern of expression in the intestinal epithelium was observed, with a gradually increasing loss of expression from crypt to tip of the villi. Occludin and claudin-1 expression were inversely correlated with Child-Pugh score (P < 0·001), the grade of oesophageal varices (P < 0·01) and endotoxin concentrations (P < 0·001). CONCLUSIONS: This study demonstrates for the first time that human liver cirrhosis induces significant alterations in enterocytes' TJs. These changes might represent an important cellular mechanism for intestinal barrier dysfunction and hyperpermeability in patients with liver cirrhosis.

Altered expression of NFY-C and RORA in colorectal adenocarcinomas.

NFY-C, a subunit of the transcription factor NFY, binds to the promoters of several eukaryotic genes, including cell cycle-related genes. RORA is a steroid hormone receptor implicated in a range of important cellular processes. We evaluated the expression of NFY-C and RORA in colorectal adenocarcinomas and normal colonic tissue. NFY-C expression was elevated in adenocarcinomas. Moreover, NFY-C mRNA levels correlated with time to disease progression, while NFY-C protein expression was significantly higher in metastatic disease. RORA expression was downregulated in CRC adenocarcinomas compared to normal controls and correlated with time to disease progression. The role of NFY-C and RORA in CRC merits further investigation.

Stimulation of oval cell and hepatocyte proliferation by exogenous bombesin and neurotensin in partially hepatectomized rats.

AIM: To investigate the effect of the neuropeptides bombesin (BBS) and neurotensin (NT) on oval cell proliferation in partially hepatectomized rats not pretreated with a known hepatocyte inhibitor. METHODS: Seventy male Wistar rats were randomly divided into five groups: I = controls, II = sham operated, III = partial hepatectomy 70% (PHx), IV = PHx + BBS (30 µg/kg per day), V = PHx + NT (300 µg/kg per day). Forty eight hours after liver resection, portal endotoxin levels and hepatic glutathione redox state were determined. α-fetoprotein (AFP) mRNA (in situ hybridisation), cytokeratin-19 and Ki67 antigen expression (immunohistochemistry) and apoptosis (TUNEL) were evaluated on liver tissue samples. Cells with morphological features of oval cells that were cytokeratin-19 (+) and AFP mRNA (+) were scored in morphometric analysis and their proliferation was recorded. In addition, the proliferation and apoptotic rates of hepatocytes were determined. RESULTS: In the control and sham operated groups, oval cells were significantly less compared to groups III, IV and V (P < 0.001). The neuropeptides BBS and NT significantly increased the proliferation of oval cells compared to group III (P < 0.001). In addition, BBS and NT induced a significant increase of hepatocyte proliferation (P < 0.001), whereas it decreased their apoptotic activity (P < 0.001) compared to group III. BBS and NT significantly decreased portal endotoxemia (P < 0.001) and increased the hepatic GSH: GSSG ratio (P < 0.05 and P < 0.001, respectively) compared to group III. CONCLUSION: BBS and NT stimulated oval cell proliferation in a model of liver regeneration, without use of concomitant suppression of hepatocyte proliferation as oval cell activation stimuli, and improved the hepatocyte regenerative response. This peptides-induced combined stimulation of oval cell and hepatocyte proliferation might serve as a possible treatment modality for several liver diseases.

Evaluation of zotarolimus-eluting metal stent in animal ureters.

BACKGROUND AND PURPOSE: Drug-eluting stents proved to minimize neointimal hyperplasia in coronary vessels. Hyperplastic reaction is the most common unwelcome event related to the use of metal mesh stents in the ureter. We evaluated the effect of zotarolimus-eluting stent (ZES) Endeavor Resolute in the porcine and rabbit ureter. MATERIALS AND METHODS: A ZES and a bare metal stent (BMS) were inserted in each ureter of 10 pigs and 6 rabbits. The insertion was performed by the retrograde approach. CT was used for the evaluation of porcine ureters while intraoperative intravenous urography (IVU) was used for rabbit ureters. The follow-up included CT or IVU every week for the following 4 weeks for pigs and 8 weeks for rabbits. Renal scintigraphies were performed before stent insertion and during the third week in all animals. Optical coherence tomography (OCT) has been used for the evaluation of the luminal and intraluminal condition of the ureters with stents. Histopathologic examination of the these ureters embedded in glycol-methacrylate was performed. RESULTS: Hyperplastic reaction was present in both stent types. BMSs in seven porcine ureters were completely obstructed while porcine ureters with ZES stents had hyperplastic tissue that did not result in obstruction. Two rabbit ureters with BMS stents were occluded while no ZES was associated with ureteral obstruction. The function of the seven porcine renal units and the two rabbit units with obstructed ureters with stents was compromised. The OCT revealed increased hyperplastic reaction in the ureters with BMS stents in comparison with those with ZESs. Although, hyperplastic reaction was present in all cases, pathologic examination revealed significantly more hyperplastic reaction in BMSs. CONCLUSION: ZESs in the pig and rabbit ureter were not related to hyperplastic reaction resulting in stent occlusion. These stents were related to significantly lower hyperplastic reaction in comparison with BMSs while inflammation rates were similar for both stent types.

Expression of the ribonucleases Drosha, Dicer, and Ago2 in colorectal carcinomas.

The pathogenesis of colorectal carcinoma (CRC) is a complex process that involves the recruitment of both genetic and epigenetic mechanisms. Recent studies underline the cardinal role of small, noncoding RNA molecules, called microRNAs (miRs), in the pathobiology of numerous physiological and pathological processes, including oncogenesis. MiR biogenesis and maturation is mainly regulated by the nuclear ribonuclease Drosha and the cytoplasmic ribonucleases Dicer and Ago2. In the present study, we investigated the expression and distribution of these molecules in three colon cancer cell lines and in human CRC samples. Drosha, Dicer, and Ago2 mRNA and protein expression was assessed with real-time PCR, western blotting, and immunofluorescence. Our experiments showed that Drosha, Dicer, and Ago2 were expressed in all the cell lines and in the majority of the CRC samples examined. The mRNA levels of Dicer were significantly augmented in stage III compared to stage II tumors. Our results suggest that Drosha, Dicer, and Ago2 are possibly implicated in CRC pathobiology and that Dicer might have a role in the progression of these tumors to advanced stages.

The survivin -31 snp in human colorectal cancer correlates with survivin splice variant expression and improved overall survival.

BACKGROUND: Survivin is involved in the regulation of cell division and survival, two key processes in cancer. The majority of studies on survivin in colorectal cancer (CRC) have focused on protein expression and less is known about the expression of survivin splicing variants or survivin gene polymorphisms in CRC. In the present study, the mRNA levels of the five known isoforms of survivin as well as survivin protein were assessed in matched normal and neoplastic colorectal tissue. Moreover, the 9386 C/T and -31 G/C polymorphisms were investigated. METHODS: Quantitative RT-PCR was used to assess mRNA levels in fresh/frozen tissue samples. Protein levels were immunohistochemically evaluated on formalin-fixed paraffin-embedded tissue sections. Individuals were genotyped using real time PCR. RESULTS: Expression of all 5 survivin splice variants as well as survivin protein was elevated in colorectal carcinomas compared to normal tissue. Specific splice variant expression differentially correlated with clinicopathological parameters. Furthermore, both snps correlated with splice variant levels or their ratios in colorectal carcinomas while the -31 G/C snp may be related to CRC development and improved overall survival. CONCLUSION: Our results support a role of survivin in colorectal carcinogenesis while the -31 G/C snp may constitute a marker of survival.

Intestinal epithelial cell proliferation, apoptosis and expression of tight junction proteins in patients with obstructive jaundice.

BACKGROUND: Intestinal hyperpermeability has been repeatedly confirmed in patients with obstructive jaundice and is considered a pivotal factor in the development of septic and renal complications in these patients. However, little is known on the mechanism(s) leading to this phenomenon. This study was undertaken to investigate the cellular and subcellular intestinal alterations in patients with obstructive jaundice. DESIGN: Sixteen patients with obstructive jaundice of malignant (n = 8, group A) or benign (n = 8, group B) aetiology, without concomitant cholangitis, and eight healthy controls (group C) were subjected to duodenal biopsy distal to the ampulla of Vater. Specimens were examined histologically and the apoptotic activity in the cryptal epithelium was recorded. Epithelial proliferation was evaluated by immunohistochemical expression of Ki67 antigen. The expression of the tight junction (TJ) proteins occludin, claudin-1, claudin-4 and claudin-7 in the intestinal epithelium was also evaluated by immunohistochemistry. RESULTS: Patients with malignant or benign obstructive jaundice presented significantly decreased intestinal epithelial cell proliferation rates compared with controls (P < 0·05), whereas no differences were detected in apoptotic activity. In a semiquantitative analysis of TJ protein expression, occludin, claudin-1 and -7 were significantly decreased (P < 0·001), whereas claudin-4 was significantly increased (P < 0·01) in jaundiced patients and their distribution was altered. No differences were detected between patients with malignant or benign obstructive jaundice for all intestinal barrier parameters studied. CONCLUSION: Decreased enterocyte proliferation and altered TJ protein expression might represent important mechanisms for intestinal barrier dysfunction and hyperpermeability in patients with extrahepatic cholestasis. The potential pharmacological modulation of these factors may lead to better control of intestinal permeability in the jaundiced patient with improved clinical outcome.

Complications and risk factors related to the extent of surgery in thyroidectomy. Results from 2,043 procedures.

OBJECTIVE: To evaluate the rate of complications and the risk factors in relation to the extent of surgery in patients undergoing thyroidectomy in a tertiary university center. DESIGN: Data were collected retrospectively from 2,043 consecutive patients who underwent thyroid surgery for various thyroid diseases at the University Hospital of Patras, Greece, between January 1996 and December 2007. Recurrent laryngeal nerve palsy (RLNP) and hypoparathyroidism were set as the primary end points, while hematoma and wound infection were set as the secondary endpoints. RESULTS: Total, near-total and subtotal thyroidectomy was performed in 1,149,777 and 117 patients, respectively. Transient RLNP occurred in 34 (1.6%) and permanent in 19 (0.9%) patients. Multivariate logistic regression analysis showed that extended resection (OR-odds ratio-1.6), Graves' disease (OR 2.7), thyroiditis (OR 2.1), recurrent goiter (OR 2.3) and thyroid malignancy (OR 1.7) were all independent risk factors for transient RLNP, whereas Graves' disease (OR 2.2) and recurrent goiter (OR 1.7) emerged as independent risk factors for permanent RLNP. The rates of transient and permanent hypoparathyroidism were 27.8% and 4.8%, respectively. Multivariate analysis for transient hypoparathyroidism revealed that the extent of surgical resection (OR 2.2), Graves' disease (OR 2.1), recurrent goiter (OR 1.7), female gender (OR 1.5) and specimen weight (OR 1.6) were independent predictors. However, the extent of surgical resection (OR 2.7), Graves' disease (OR 1.8), recurrent goiter (OR 1.5) and malignant disease (OR 1.5) were independent risk factors for permanent hypoparathyroidism. Postoperative wound infection and hematoma occurred in 6 (0.3%) and 27 (1.3%) patients, respectively. No correlation was observed between wound infection or postoperative hemorrhage and the extent of surgery. CONCLUSIONS: Despite the higher morbidity, total thyroidectomy is emerging as an attractive surgical option even for benign thyroid disease due to the risk of subclinical (occult) malignancy, the possibility of goiter relapse as well as of the increased risk of complications following reoperation.