Comparison of different tissue sampling methods for protein extraction from formalin-fixed and paraffin-embedded tissue specimens.

AIMS: Protein extracts from formalin-fixed and paraffin-embedded (FFPE) tissue for proteomic analysis has recently gained attention. In this study, we explored the possibility to standardize tissue sampling from paraffin blocks and compared the protein extracts with those obtained from fresh frozen material. MATERIALS AND METHODS: Fresh frozen and FFPE material was obtained from five patients with pancreatic ductal adenocarcinoma either by cutting sections with a microtome or by stamping a cylinder with tissue micro-array technology. All samples were weighed, forwarded to protein extraction and analyzed by polyacrylamide gel electrophoresis and Western blotting. Immunohistochemistry allocated proteins in tissue sections. RESULTS: Sampling of tissue was highly reproducible, as assessed by sample weight. While protein concentrations were significantly higher in fresh frozen material compared to FFPE material, equal amounts of protein were extracted from FFPE using either paraffin sections or core cylinders in SDS-PAGE, all three procedures showed comparable protein patterns. In Western blotting, annexin I had the same molecular weight independent of the sample source and sampling procedure. CONCLUSIONS: The sampling of FFPE specimens for protein extraction and analysis can be standardized, uncovering equal amounts of tissue and protein. In addition, the proteins extracted from FFPE tissue seem to be the same compared with those extracted from fresh frozen tissue.

Angiotensin I-converting enzyme (CD143) is down-regulated in focal nodular hyperplasia of the liver.

In surgical pathology, focal nodular hyperplasia (FNH) is sometimes difficult to diagnose, and liver cirrhosis (LC) and hepatocellular adenoma (HA) are often among the differential diagnoses. Recently, we found a reduced expression of angiotensin I-converting enzyme (CD143) in FNH compared with cirrhotic and noncirrhotic liver. Intrigued by this observation, we investigated the expression pattern of CD143 in FNH, LC, and HA and its possible diagnostic value. The expression of CD143 was studied by immunohistochemistry in 20 FNHs, 13 corresponding extralesional noncirrhotic liver parenchyma, 20 patients with LC, and four HAs. Endothelial cells were identified with antibodies directed against CD31 and CD34. CD31+, CD34+, and CD143+ sinusoidal endothelial cells were found in extralesional liver, LC, HA, and FNH. However, the number of CD143+ sinusoidal endothelial cells and the intensity of immunostaining were significantly reduced in FNH compared with extralesional liver, LC, and HA. The expression of CD143 was further assessed using a numerical scoring system ranging from 0 to 6. The mean immunoreactivity score for CD143 was 2.4 +/- 1.7 for sinusoidal endothelial cells in FNH, 5.7 +/- 0.6 in extralesional liver, 4.8 +/- 1.1 in LC, and 5.8 +/- 0.5 in HA. The differences between the mean immunoreactivity scores for CD143 were highly significant. The difference between FNH and extralesional liver was confirmed on transcriptional level by fluorescence-mediated real-time RT-PCR, which also showed a significantly decreased level of CD143 mRNA in FNH. Our study provides evidence that CD143 is down-regulated in FNHs and that the phenotype of endothelial cells lining the sinusoids in FNH differs from those in non-neoplastic liver, LC, and HA. The observed variations in expression patterns for CD143 might be of diagnostic use in surgical pathology.

Ectopeptidases are differentially expressed in hepatocellular carcinomas.

We investigated the expression pattern of neprilysin (CD10), aminopeptidase N (CD13) and angiotensin-I converting enzyme (CD143) in hepatocellular carcinomas (HCC), and their putative roles in hepatocarcinogenesis. Tissue samples were obtained from 31 patients with HCC. Tissue samples obtained from non-neoplastic liver, fetal livers and focal nodular hyperplasias (FNH) were used by comparison. Transcription and expression of CD10, CD13, and CD143 were studied by quantitative RT-PCR, Western blotting, and immunohistochemistry. Cell proliferation assays were performed with the C3A hepatoma cell line. The mRNA and protein of each of CD10, CD13 and CD143 were differentially expressed in HCCs. CD10 was decreased in HCCs as compared to non-neoplastic liver tissue, while CD13 and CD143 were mildly increased. In fetal liver and FNHs, the expression of CD10 was less intense than in the surrounding non-tumorous liver. The expression patterns of CD13 and CD143 in fetal livers and FNHs were similar to HCCs and were predominantly localized in bile canaliculi (CD13) and endothelial cells (CD143). CD10 and CD13 mRNAs were expressed by C3A cells and blocking either CD10 or CD13 ectopeptidase activity retarded cell growth significantly in vitro. We demonstrate that ectopeptidases are differentially expressed in HCCs and may have influence on tumor biology. Overall, expression of CD10 in non-neoplastic and neoplastic hepatocytes appears to correlate inversely with their state of proliferation or differentiation. CD13 shows a characteristic canalicular distribution pattern and may be important for cell polarization and bile compartmentalization in HCCs, while CD143 may influence angiogenesis.

Irrelevance of microsatellite instability in the epidemiology of sporadic pancreatic ductal adenocarcinoma.

BACKGROUND AND AIMS: Pancreatic cancer risk is increased in Lynch syndrome (LS) patients with mismatch repair gene defects predisposing to colonic and extracolonic cancers with microsatellite instability (MSI). However, the frequency of MSI pancreatic cancers has never been ascertained in consecutive, unselected clinical series, and their contribution to the sporadic and inherited burden of pancreatic cancer remains to be established. Aims of the study were to determine the prevalence of MSI in surgically resected pancreatic cancers in a multicentric, retrospective study, and to assess the occurrence of pancreatic cancer in LS. METHODS: MS-status was screened by a panel of 5 mononucleotide repeats (Bat26, Bat25, NR-21, NR-24 and NR-27) in 338 consecutive pancreatic ductal adenocarcinoma (PDAC), resected at two Italian and one German referral centres. The personal history of pancreatic cancer was assessed in an independent set of 58 probands with LS and in 138 first degree relatives who had cancers. RESULTS: Only one PDAC (0.3%) showed MSI. This was a medullary type cancer, with hMLH1-deficiency, and no identified germ-line mutation but methylation of hMLH1. Pancreatic cancer occurred in 5 (2.5%) LS patients. Histological sampling was available for 2 cases, revealing PDAC in one case and an ampullary cancer in the other one. CONCLUSIONS: MSI prevalence is negligible in sporadic, resected PDAC. Differently, the prevalence of pancreatic cancer is 2.5% in LS patients, and cancers other than PDAC may be encountered in this setting. Surveillance for pancreatic cancer should be advised in LS mutation carriers at referral centers.

Angiotensin II-generating enzymes, angiotensin-converting enzyme (ACE) and mast cell chymase (CMA1), in gastric inflammation may be regulated by H. pylori and associated cytokines.

BACKGROUND: The local angiotensin II system (LAS) has numerous functions, including the regulation of growth and differentiation in the gastrointestinal tract. Angiotensin II (AngII) may be generated by angiotensin-I-converting enzyme (ACE) or mast cell chymase (CMA1) and plays an important role in inflammatory processes, although opinions differ as to which AngII-generating enzyme is primarily associated with AngII-mediated effects. ACE inhibitors have been shown to have a protective or healing effect on gastric ulcers and colitis in animal models, which could be related to the local expression of ACE. METHODS: The localisation of ACE and CMA1 was examined immunohistochemically in Helicobacter pylori gastritis, non-H. pylori gastritis, gastric ulcers and non-lesional gastric tissues. Using real-time qRT-PCR, ACE- and CMA1-mRNA expression in gastric cell lines were examined and changes in ACE levels after exposure to H. pylori or cytokines (IL-1beta, IL-6, IL-8, TNF, TGFbeta1) were quantified. RESULTS: ACE and CMA1 were not expressed in the non-lesional foveolar epithelium. Cytoplasmic staining for ACE in fundic chief cells, and apical membranous expression of ACE in the mucin-secreting cells of the antral and pyloric region was observed. ACE was found in endothelial cells of the gastric ulcer granulation tissue and CMA1 was strongly expressed in mast cells. ACE but not CMA1 was expressed in the MKN28, N87 and MKN45 gastric cell lines, and ACE mRNA expression was regulated by both H. pylori and the cytokines. CONCLUSIONS: ACE in the gastric mucosa and the microvasculature of granulation tissue may represent a novel therapeutic target for the promotion of healing processes in gastritis and ulceration using ACE inhibitors or AT1R antagonists.

Proteomics of pancreatic cancer.

Pancreatic cancer is a devastating disease, with a mortality rate almost identical with its incidence. Late diagnosis and limited therapeutic options make early detection of pancreatic cancer a pressing clinical problem. In this context, the investigation of the pancreatic cancer proteome has recently gained considerable attention because profiles of proteins may be able to more accurately identify disease states, such as cancer. Recent pancreatic cancer proteome studies may be categorized into basic studies cataloguing the pancreatic proteome, studies investigating differential protein expression patterns, and studies searching for proteome-based biomarkers for early cancer detection and differentiation. Although these studies clearly demonstrate that a range of biological samples are suitable for proteomic analyses, comparison of different studies is problematic due to the diversity of methodologies, sample sources, and characterization of patient populations. Reproducibility between studies has rarely been investigated, and no investigation has compared the different methods of proteomic research. The results of this review have shown that more stringent requirements concerning the design and the analysis of future studies should be implemented. These include an adequate patient number, obligatory histological examination of tissues, appropriate control groups, identification of proteins and peaks, validation of differential expression using independent cohorts and/or a second methodology, and, finally, demonstration of result reproducibility. This will hopefully lead to the discovery of prognostic and predictive biomarkers that help to improve prognosis of pancreatic cancer patients.