Keratin 7 expression in hepatic cholestatic diseases.

We evaluated keratin 7 (K7) hepatocellular expression in 92 patients with common types of acute and chronic cholestatic diseases caused by bile duct obstruction/destruction or parenchymal lesions [acute hepatitis (n=20), mixed/pure cholestasis (n=16), primary biliary cholangitis-PBC (n=35), primary sclerosing cholangitis-PSC (n=10), vanishing bile duct syndrome (n=3), complete large bile duct obstruction due to space-occupying lesions (n=8)]. K7 immunohistochemical hepatocellular expression and ductular reaction (DR) were semi-quantitatively assessed. Results were correlated with liver enzyme serum levels, cholestasis type, histological features, hepatocellular Ki67 labelling index (LI) and HepPar1 expression. Hepatocellular K7 expression was detected in 87% (81/92) cases and in all cholestatic disease types with lowest incidence in pure/mixed cholestasis and highest in incomplete bile duct obstruction (iBDO), reaching 100% in PSC. K7-positive hepatocytes had low Ki67 LI (0-5%) retaining HepPar1 expression, irrespective of disease type. PSC cases had high K7 hepatocellular expression even with intact bile ducts, a feature that may aid differential diagnosis of cholestatic syndromes. K7 hepatocellular expression significantly correlated with cholestasis type, bile duct loss and fibrosis stage. It was higher in milder acute cholestatic hepatitis showing inverse correlation with hepatocyte proliferation and serum transaminase levels. In iBDO, younger age independently correlated with high K7 expression, while serum GGT levels showed a nearly significant correlation. Correlation with DR findings implied that K7-positive hepatocytes may result through metaplasia. In conclusion, K7 hepatocellular expression is a sensitive though non-specific marker of cholestasis. It may represent a cytoprotective reaction of resting hepatocytes in cholestasis of longer duration especially in younger patients.

NFκB1 is a suppressor of neutrophil-driven hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) develops on the background of chronic hepatitis. Leukocytes found within the HCC microenvironment are implicated as regulators of tumour growth. We show that diethylnitrosamine (DEN)-induced murine HCC is attenuated by antibody-mediated depletion of hepatic neutrophils, the latter stimulating hepatocellular ROS and telomere DNA damage. We additionally report a previously unappreciated tumour suppressor function for hepatocellular nfkb1 operating via p50:p50 dimers and the co-repressor HDAC1. These anti-inflammatory proteins combine to transcriptionally repress hepatic expression of a S100A8/9, CXCL1 and CXCL2 neutrophil chemokine network. Loss of nfkb1 promotes ageing-associated chronic liver disease (CLD), characterized by steatosis, neutrophillia, fibrosis, hepatocyte telomere damage and HCC. Nfkb1(S340A/S340A)mice carrying a mutation designed to selectively disrupt p50:p50:HDAC1 complexes are more susceptible to HCC; by contrast, mice lacking S100A9 express reduced neutrophil chemokines and are protected from HCC. Inhibiting neutrophil accumulation in CLD or targeting their tumour-promoting activities may offer therapeutic opportunities in HCC.

Liver biopsy in alcoholic and non-alcoholic steatohepatitis patients.

Alcoholic liver disease and non-alcoholic liver disease share a similar histological spectrum that starts with 'simple' steatosis, and may be accompanied by inflammation. Alcoholic steatohepatitis and non-alcoholic steatohepatitis (NASH) are progressive forms of alcoholic liver disease and non-alcoholic liver disease, respectively, and can evolve into cirrhosis. The currently accepted minimum diagnostic criteria for steatohepatitis include steatosis, lobular inflammation and hepatocellular injury, but not fibrosis. Steatosis involving more than 5% of hepatocytes is required for the diagnosis of non-alcoholic fatty liver disease, but is not necessary for the diagnosis of alcoholic liver disease. Lobular inflammation is usually mild and frequently consists of a mixed, acute and chronic, inflammatory cell infiltrate composed of neutrophils and mononuclear cells. The presence of large numbers of neutrophils favors an alcoholic etiology. Hepatocellular injury in fatty liver disease usually occurs in the form of ballooning, but it can also present as apoptotic (acidophilic) bodies and lytic necrosis. The characteristic pattern of fibrosis in non-cirrhotic steatohepatitis is pericellular/perisinusoidal and is the result of deposition of collagen in the space of Disse. In both alcoholic steatohepatitis and NASH, sinusoidal collagen formation is the result of hepatic stellate cell activation that, in NASH, has been correlated with the grade of steatosis and fibrosis.

Right sited renal cell carcinoma metastasizing to the contralateral ovary: case report and review of the literature.

Ovarian metastases from renal cell carcinoma are rare, with only 22 cases reported in the literature. We report a case of a 45-year-old woman, who developed left ovarian and right adrenal metastases 3 months after diagnosis of clear cell renal cell carcinoma and review the literature. This is the fourth reported case of right renal cell carcinoma metastasizing to the left ovary. The patient is alive 4 years after resection of the ovarian tumor, treated with sunitinib. We conclude that, although rare, metastatic renal cell carcinoma should be included in the differential diagnosis of ovarian tumors with clear cell histology.

Diagnostic value of rapid urease test and urea breath test for Helicobacter pylori detection in patients with Billroth II gastrectomy: a prospective controlled trial.

AIM: The aim of this work was to assess the reliability of rapid urease test (RUT) and urea breath test (UBT) for detecting Helicobacter pylori (H. pylori) in patients with Billroth II (BII) gastrectomy, using histology as reference. METHODS: In this prospective controlled study, 31 consecutive patients with BII gastrectomy and 73 controls who had an indication for endoscopy were included. Their H. pylori status was assessed with biopsies for histology, RUT and UBT. Histology served as the gold standard. Only the biopsies from the gastric fundus were evaluated. Specificity, sensitivity, positive and negative predictive value, degree of agreement and k-statistics were used. RESULTS: RUT and UBT for detecting H. pylori in the control group had excellent agreement [97%, kappa (k)=0.94 and 99%, k=0.97 respectively] with biopsies. In BII patients, RUT from fundic biopsies had very good agreement (87%, k=0.74) compared to histology from fundic biopsies, whereas the UBT was unreliable (agreement: 71%, k=0.41) compared to histology. CONCLUSION: The RUT from fundic biopsies in BII patients is a reliable test for H. pylori detection, whereas the UBT is unreliable.

Metabolic syndrome is associated with severe fibrosis in chronic viral hepatitis and non-alcoholic steatohepatitis.

BACKGROUND: The prevalence of metabolic syndrome and its possible impact on the severity of liver histological lesions have not been studied prospectively in chronic liver diseases. AIM: To investigate the prevalence of metabolic syndrome in patients with chronic viral hepatitis or non-alcoholic steatohepatitis, and to determine its associations with histological severity. METHODS: We prospectively included 317 patients (hepatitis B e antigen-negative chronic hepatitis B: 95, chronic hepatitis C: 176, non-alcoholic steatohepatitis: 46) with liver biopsy. Metabolic syndrome was defined using the Adult Treatment Panel III criteria. Histological lesions were evaluated according to Ishak's or Brunt's classification. RESULTS: Metabolic syndrome was present in 10.4% of patients being significantly more prevalent in non-alcoholic steatohepatitis than in chronic viral hepatitis (41.3% vs. 5.1%, P < 0.001). In chronic viral hepatitis, cirrhosis (stages 5-6) was independently associated with increasing age, higher aspartate aminotransferase and gamma-glutamyl-transpeptidase levels, severe necroinflammation and metabolic syndrome (P = 0.016). In non-alcoholic steatohepatitis, severe fibrosis (stages 3-4) was independently associated with severe necroinflammation and metabolic syndrome (P = 0.033). Presence of metabolic syndrome was not associated with presence or severity of steatosis both in chronic viral hepatitis and in non-alcoholic steatohepatitis. CONCLUSION: Metabolic syndrome is more prevalent in non-alcoholic steatohepatitis than in chronic viral hepatitis; it is associated independently with more severe fibrosis but not with the severity of steatosis, both in chronic viral hepatitis and in non-alcoholic steatohepatitis.

Serum apoptotic caspase activity as a marker of severity in HBeAg-negative chronic hepatitis B virus infection.

BACKGROUND AND AIMS: In chronic hepatitis C and non-alcoholic fatty liver disease, apoptotic caspases are activated in liver, and serum caspase activity has been suggested as a sensitive marker of early liver injury. An investigation was carried out into whether the serum levels of caspase-generated fragments of cytokeratin-18 (CK-18) are associated with the severity of liver lesions in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B virus (HBV) infection. Patients/ METHODS: CK-18 fragment serum levels were determined in 115 treatment-naive, consecutive HBV patients and 30 healthy controls. Hepatic-expression of CK-18 fragments was evaluated by immunocytochemistry in chronic hepatitis B patients. RESULTS: CK-18 fragment levels (U/l) were significantly lower in healthy controls (mean (SD), 154 (31)) than in 53 inactive carriers (172 (24), p = 0.003) and in 62 chronic hepatitis B patients (474 (488), p<0.001). The receiver operating characteristic curve showed excellent diagnostic accuracy (c-statistic: 0.87) for differentiating inactive carriers from chronic hepatitis B patients. A CK-18 fragment cut-off level of 240 U/l gave a sensitivity of 60%, and a specificity and positive predictive value of 100% for chronic hepatitis B diagnosis. CK-18 fragment levels were also lower in inactive carriers than in 16 chronic hepatitis B patients with transiently normal alanine aminotransferase (ALT; 327 (256), p = 0.001), offering good accuracy for such a differentiation (c-statistic: 0.78). In chronic hepatitis B patients, serum CK-18 fragments correlated positively with ALT/aspartate aminotransferase (AST), viraemia, grading score and their immunohistochemical hepatic expression, and negatively with platelet counts, but not with fibrosis or steatosis severity. CONCLUSIONS: Serum apoptotic caspase activity is strongly associated with the presence of liver injury in patients with HBeAg-negative chronic HBV infection. CK-18 fragment levels seem to be a very useful marker for differentiation between the inactive HBV carrier state and HBeAg-negative chronic hepatitis B, but not for estimation of the severity of liver histological lesions among HBeAg-negative chronic hepatitis B patients.

Gene amplification is a relatively frequent event leading to ZBTB7A (Pokemon) overexpression in non-small cell lung cancer.

ZBTB7A (Pokemon) is a member of the POK family of transcriptional repressors. Its main function is the suppression of the p14ARF tumour suppressor gene. Although ZBTB7A expression has been found to be increased in various types of lymphoma, there are no reports dealing with its expression in solid tumours. Given that p14(ARF) inhibits MDM2, the main negative regulator of p53, we hypothesized that overexpression of ZBTB7A could lead indirectly to p53 inactivation. To this end, we examined the status of ZBTB7A and its relationship with tumour kinetics (proliferation and apoptosis) and nodal members of the p53 network in a panel of 83 non-small cell lung carcinomas (NSCLCs). We observed, in the majority of the samples, prominent expression of ZBTB7A in the cancerous areas compared to negligible presence in the adjacent normal tissue elements. Gene amplification (two- to five-fold) was found in 27.7% of the cases, denoting its significance as a mechanism driving ZBTB7A overproduction in NSCLCs. In the remaining non-amplified group of carcinomas, analysis of the mRNA and protein expression patterns suggested that deregulation at the transcriptional and post-translational level accounts for ZBTB7A overexpression. Proliferation was associated with ZBTB7A expression (p = 0.033) but not apoptosis. The association with proliferation was reflected in the positive correlation between ZBTB7A expression and tumour size (p = 0.018). The overexpression of ZBTB7A in both p53 mutant and p53 wild-type cases, implies either a synergistic effect or that ZBTB7A exerts its oncogenic properties independently of the p14(ARF)-MDM2-p53 axis. The concomitant expression of ZBTB7A with p14(ARF) (p = 0.039), instead of the anticipated inverse relation, supports the latter notion. In conclusion, regardless of the pathway followed, the distinct expression of ZBTB7A in cancerous areas and the association with proliferation and tumour size pinpoints a role for this novel cell cycle regulator in the pathogenesis of lung cancer.

Co-expression of trypsin and tumour-associated trypsin inhibitor (TATI) in colorectal adenocarcinomas.

Trypsin and its specific inhibitor, TATI (tumour-associated trypsin inhibitor), are expressed in normal human pancreas and in a variety of tumours. The aim of the present study was to assess the parallel expression of trypsin and TATI in colorectal cancer, in comparison with their expression in normal epithelial tissue, since proteases and their inhibitors are thought to be co-expressed in malignant neoplasms. We also assessed the possible significance of their expression as a means of differentiation between normal and malignant tissue. We examined qualitatively and semi-quantitatively the immunohistochemical expression of trypsin and TATI on paraffin-embedded serial tissue sections from 91 colorectal adenocarcinomas. The reverse-transcriptase-polymerase-chain reaction (RT-PCR) was also performed on fresh malignant tissue from 55 of the above adenocarcinomas. Normal and non-malignant tissues adjacent to the tumours were also evaluated. Cytoplasmic expression of trypsin (more than 25% of the cancer cells positive) was found in 67 (73.6%) adenocarcinomas, whereas TATI was expressed in the cytoplasm of 59 (64.8%) cases studied. Statistical analysis using Spearman's test has demonstrated a significant correlation between trypsin and TATI immunohistochemical expression (p<0.01). RT-PCR showed co-expression of trypsin and TATI mRNA in all carcinomas studied. Distinct patterns of trypsin and TATI immunohistochemical expression were observed in adjacent, non-malignant tissues, where both trypsin and TATI mRNA were also detected. Normal tissues were negative by immunohistochemistry. Our results indicate co-expression of trypsin and TATI in colorectal tumours both at the mRNA and protein level. We conclude that in colorectal neoplasms, high levels of trypsin and TATI may be important for malignant tumour formation and/or metastatic process.

Recent advances in early prostatic cancer.

Early prostatic carcinoma is a slowly progressing, localized malignant tumor which has been recently discovered with increased frequency due to the use of improved diagnostic methods. The combination of digital rectal examination, serum PSA level and transrectal ultrasound is currently the best available diagnostic tool, although other putative diagnostic markers and techniques are being investigated. Core needle biopsy may follow if there is suspicion of malignancy and in doubtful cases the most useful antibody for the immunohistochemical diagnosis of early, low grade prostatic carcinoma is clone 34 beta E12. Cytogenetic techniques and molecular biological methods are increasingly being used for further investigating localized prostate carcinomas in order to identify early molecular targets and alterations, which may lead to progression. Chromosome abnormalities, cell to cell and cell to matrix interactions, changes in the status of steroid hormone receptors, oncogenes and tumor suppressor genes, as well as other, as yet unclear, events may be of importance in prostate carcinogenesis and the progression of early malignant tumors to aggressive phenotypes. A variety of putative prognostic markers, apart from serum PSA levels, histological grade and tumor volume, such as neuroendocrine differentiation, angiogenesis, cell proliferation labeling index and ploidy analysis may prove useful in evaluating tumor progression in early prostatic carcinomas. The final and most important goal of all investigations related to early prostate cancer is to contribute to the best therapeutic management of the individual patient.

Proliferating cell nuclear antigen and Ki-67 labeling in hepatocellular nodules: a comparative study.

AIMS/BACKGROUND: The morphologic differential diagnosis of hepatocellular nodules (HCN) is frequently difficult and objective criteria would be useful in the categorization of such lesions. This study evaluated the proliferative activity of HCN, including regenerative, macroregenerative (MRN), cirrhotic, dysplastic, and hepatocellular carcinoma (HCC), as well as intranodular cytologic changes such as bile-stained hepatocytes, eosinophilia, clear, large cell (LCC) and small cell (SCC) change, by comparing the cellular density (CD), labeling indices (LI) and density (DP) of two proliferation markers. METHODS: Routinely processed tissue sections from 45 HCN from 17 adult liver explants were studied by immunohistochemistry for PCNA and Ki-67 (MIB-1). RESULTS: A progressive increase in LI from regenerative to dysplastic nodules to HCC was observed with both proliferation markers. The values of the two markers were significantly correlated (p<0.001). CD, PCNA and MIB-1 LI and DP values were significantly lower in regenerative compared to dysplastic nodules or HCC. MRNs had lower PCNA and MIB-1 LI and DP than regenerative nodules, but similar CD. There were no statistically significant differences in CD, PCNA, and MIB-1 LI and DP between dysplastic nodules and HCC, comparing high versus low grade dysplasia, or HCC smaller than 2 cm with those larger than 2 cm. The CD and proliferation indices LI and DP were higher in HCC than in the surrounding non-neoplastic parenchyma. Lesions with clear cell, eosinophilic and large cell change had CD, PCNA and MIB-1 indices similar to those of regenerative nodules, while these were lower in bile-stained hepatocellular lesions (p<0.01). SCC showed CD, PCNA and MIB-1 LI and DP similar to HCC and higher than surrounding regenerative lesions (p<0.003). CONCLUSIONS: Our results suggest that PCNA and MIB-1 values are closely correlated in HCN. Regenerative nodules are characterized by low cellular proliferation, while dysplastic nodules are usually highly proliferative lesions and may represent an early stage in hepatocarcinogenesis. Hepatocellular lesions characterized by bile stained hepatocytes, eosinophilic, clear and large cell change have low proliferation rates and may not be significant for the development of malignancy.

Osteopontin expression in ovarian carcinomas and tumors of low malignant potential (LMP).

Expression of osteopontin (OPN) by ovarian tumors is not known. Neoplasms arising from the ovarian epithelium are distinguished in adenocarcinomas and borderline tumors (LMPs), which overall have a favorable prognosis even with omental implants. Tissues from primary ovarian tumors and their metastases from 30 patients (16 LMPs and 14 adenocarcinomas) were evaluated for OPN expression by immunohistochemistry, Western blotting, and in situ hybridization. OPN was weak or absent in 93% of ovarian adenocarcinomas or their metastases. In contrast, 81.5% of the LMPs and 50% of omental and lymph node implants were OPN positive (P < .028). Histological type, grade, or clinical stage did not correlate with OPN expression. Expression of OPN primarily by ovarian neoplasms with favorable prognosis is an intriguing new finding of potential importance in the pathogenesis of ovarian LMPs.

Role of neutrophils in hepatotoxicity induced by oral acetaminophen administration in rats.

Acetaminophen (APAP) is a common analgesic and antipyretic compound which, when administered in high doses, has been associated with significant morbidity and mortality, secondary to hepatic toxicity. To date, the mechanism(s) whereby APAP induces liver injury remains to be delineated. This study investigated the potential role of neutrophils as contributors to liver injury in rats administered sublethal doses of APAP. Oral APAP administration (650 mg/kg) was associated with increases in serum alanine transaminase (ALT) levels indicating biochemical evidence of significant liver damage. Furthermore, histological analyses verified significant hepatocellular necrosis as well as enhanced myeloperoxidase staining in these liver specimens. However, if animals were pretreated with antineutrophil sera prior to APAP administration, neutrophil counts remained depressed, ALT levels were significantly decreased, and the degree of liver injury was attenuated on a histological level. Taken together these data suggest that neutrophils mediate, at least in part, the hepatotoxic effects of oral acetaminophen administration in rats.

Retinoblastoma protein in human breast carcinoma: immunohistochemical study using a new monoclonal antibody effective on routinely processed tissues.

Cyclic phosphorylation/dephosphorylation of the retinoblastoma gene product (pRB) has been found to play a central role in the progression of the normal cell cycle, through modulation of the activity of the E2F family of transcription factors. Mutations of the retinoblastoma gene have been described in a wide variety of human malignancies including carcinomas of the breast. The present investigation reports the production and application of a new monoclonal antibody in an immunohistochemical study of pRB expression in 233 primary breast carcinomas, allowing an assessment of the contribution made by this tumour suppressor gene to tumour development and progression. Overall, there was loss of pRB expression in 21 per cent of breast tumours. Although high-grade tumours were found to lack detectable pRB more frequently than low-grade tumours, the difference did not prove statistically significant. In addition, pRB immunostaining was not related significantly to relapse or survival. No significant correlations were observed between apparent loss of pRB and tumour size, parity, patient lymph-node status, p53, c-erbB-2, c-jun, EGFR or steroid hormone receptor expression. Preliminary findings, however, did suggest a relationship between pRB expression and response to endocrine therapy.

Innervation of the liver: morphology and function.

Although it has been known for many years that the liver receives a nerve supply, it is only with the advent of immunohistochemistry that this innervation has been analysed in depth. It is now appreciated not only that many different nerve types are present, but also that there are significant differences between species, especially in the degree of parenchymal innervation. This has stimulated more detailed investigation of the innervation of the human liver in both health and disease. At the same time, functional studies have been underlining the important roles that these nerves play in processes as diverse as osmoreception and liver regeneration. This article briefly reviews current understanding of the morphology and functions of the hepatic nerve supply.

c-erb B-2 and p53 expression in breast cancer fine needle aspirates.

The aim of this study was to co-evaluate c-erb B-2 and p53 protein expression in breast cancer fine needle aspirates (FNA) and to compare this with histological variables and the immunohistochemical phenotype of the tumours. Furthermore, we assessed the relationship of c-erb B-2 and p53 immunocytochemical expression to tumour prognostic factors. We examined 124 breast cancer FNAs and 79 matched surgical specimens using the avidin-biotin complex (ABC) and the alkaline phosphatase immunocytochemical techniques. C-erb B-2 immunopositivity was detected in 37.9% of the FNAs, while 31.7% were positive for p53. A statistically significant correlation was observed between p53 negativity and absence of c-erb B-2 immunostaining in the FNAs (P = 0.0007). Smears from infiltrating ductal carcinomas tended to be more frequently positive for p53 (36.7%) than those from lobular carcinomas (11.7%) (P = 0.054). In matched tumour tissues, c-erb B-2 was positive in 16.7% and p53 in 19% of cases. The immunocytochemical results for both c-erb B-2 and p53 were significantly correlated with the immunohistochemical results. There was no correlation between c-erb B-2 and p53 immunostaining, in both FNAs and tissues, and patients menopausal status, tumour size, grade and lymph node status.

p53 protein as a prognostic indicator in breast carcinoma: a comparison of four antibodies for immunohistochemistry.

We examined the reactivity of four p53-specific monoclonal antibodies--PAb 1801, p53-BP-12, D07 and CM1--on sections of formalin-fixed tissue collected from 245 breast carcinomas. Immunodetection of p53 varied between 37.6% and 46.6%. The greatest variation was observed among lobular carcinomas and low-grade tumors in which immunodetection varied between 8.3% and 27.3%. In contrast, immunodetection of p53 in invasive ductal carcinomas was subject to a lower degree of variability with between 40.6% and 49.7% of these tumours proving to be positive. In general, we found antibodies PAb 1801 and DO7 to be the most effective in immunolocalising p53. Immunodetection of p53 with each of the four antibodies was found to correlate strongly with tumour grade. In survival analysis, the results gained using antibody PAb 1801 proved to be of greatest statistical significance and to provide the strongest index of prognosis. A significant relationship was observed between immunodetection of p53 with each of the four antibodies and poor responsiveness to endocrine therapy. In addition, relationships were also observed between p53 immunostaining and tumour oestrogen receptor (ER) status as well as c-jun expression. We observed no correlation between abnormalities of the p53 and the Rb gene products or between elevated c-erbB-2 or epidermal growth factor receptor (EGFR) expression and immunodetection of p53.

p53 immunostaining as a marker of malignancy in cytologic preparations of body fluids.

The accurate identification of suspicious cells in cytologic preparations is a common problem in diagnostic cytopathology. Recent studies have shown that mutation of the p53 gene may be the most common genetic event in human malignancy. Mutation leads to altered conformation and increased half-life of the p53 protein, resulting in detectability by immunocytochemistry. The usefulness of p53 immunocytochemical staining as a marker of malignancy in the cytologic analysis of body fluids was investigated in the present study. One hundred fifty-four serial samples of body fluids submitted for cytologic diagnosis were also examined for p53 immunoreactivity. Of 121 cases reported as cytologically benign, 3 (2.5%) stained positively for p53; 16 samples were cytologically malignant, and 7 (43.7%) of these were positive for p53 (P < .001). Of those reported as suspicious but not conclusively malignant, 4 of 17 (23.5%) showed p53 immunoreactivity. On review, two of the three patients whose samples were benign cytologically yet showed positive p53 staining had histologic evidence of malignancy. The third patient died without a postmortem examination. Of the 17 cytologically suspicious cases, 16 (94.1%) were later proven to be malignant, and p53 was positive in 4 (25%). These results suggest that p53 immunostaining could be of value as a marker of malignancy in the cytologic examination of body fluids. The presence of p53 immunoreactivity in cytologic samples is strongly suggestive of malignancy, though its absence does not exclude neoplasia.