Immunohistochemical detection of polyductin and co-localization with liver progenitor cell markers during normal and abnormal development of the intrahepatic biliary system and in adult hepatobiliary carcinomas.

The longest open reading frame of PKHD1 (polycystic kidney and hepatic disease 1), the autosomal recessive polycystic kidney disease (ARPKD) gene, encodes a single-pass, integral membrane protein named polyductin or fibrocystin. A fusion protein comprising its intracellular C-terminus, FP2, was previously used to raise a polyclonal antiserum shown to detect polyductin in several human tissues, including liver. In the current study, we aimed to investigate by immunohistochemistry the detailed polyductin localization pattern in normal (ductal plate [DP], remodelling ductal plate [RDP], remodelled bile ducts) and abnormal development of the primitive intrahepatic biliary system, known as ductal plate malformation (DPM). This work also included the characterization of polyductin expression profile in various histological forms of neonatal and infantile cholestasis, and in cholangiocellular carcinoma (CCC) and hepatocellular carcinoma (HCC). We detected polyductin expression in the intrahepatic biliary system during the DP and the RDP stages as well as in DPM. No specific staining was found at the stage of remodelled bile ducts. Polyductin was also detected in liver biopsies with neonatal cholestasis, including mainly biliary atresia and neonatal hepatitis with ductular reaction as well as congenital hepatic fibrosis. In addition, polyductin was present in CCC, whereas it was absent in HCC. Polyductin was also co-localized in some DP cells together with oval stem cell markers. These results represent the first systematic study of polyductin expression in human pathologies associated with abnormal development of intrahepatic biliary tree, and support the following conclusions: (i) polyductin expression mirrors developmental properties of the primitive intrahepatic biliary system; (ii) polyductin is re-expressed in pathological conditions associated with DPM and (iii) polyductin might be a potential marker to distinguish CCC from HCC.

Bile duct to portal space ratio and ductal plate remnants in liver disease of infants aged less than 1 year.

AIM: To validate the bile duct to portal space ratio as an independent factor useful for the prognosis of neonatal liver disease. METHODS: We assessed the maturation of the intrahepatic bile duct system (IBDS) in 87 consecutive infants aged less than 1 year undergoing non-subcapsular, adequate (at least six portal tracts), liver needle biopsies because of hepatomegaly and/or cholestasis. The maturation of the IBDS was evaluated by immunohistochemistry with an antibody directed to cytokeratin 7 (CK7), a biliary-type intermediate filament of the cytoskeleton, and a schema showing the IBDS remodelling. We used five categories to fit the different patterns of the IBDS remodelling using the ratio between the number of bile ducts and the number of portal tracts (BD/PT) and the presence of abnormal reaction patterns (marked intra-acinar pseudorosettes and/or periportal ductular proliferation): (A) abnormal reaction patterns with any BD/PT; (B) BD/PT = 0; (C) 0.1 < or = BD/PT < 0.5; (D) 0.5 < or = BD/PT < 0.9; and (E) BD/PT > 0.9 (B-E categories: no abnormal reaction patterns). Further, we evaluated cholestasis, portal fibrosis (PF), portal inflammation (PI), giant cell transformation (GCT), and extramedullary haematopoiesis (EMH). RESULTS: We identified A-E categories in 24, 14, 17, 8, and 24 biopsies, respectively. B and C categories were frequently observed in biliary atresia (BA), A category in neonatal hepatitis (NH), A-C categories in paucity of intrahepatic bile ducts (PIBD), and E category in 'other liver diseases' (OLD). Cholestasis, PI, GCT, and EMH were more frequent in A and C, while PF was variably seen in all categories. The lowest survival rate occurred in B (Kaplan-Meier estimator). CONCLUSIONS: (1) Biliary epithelial cell patterns recapitulate the primitive stages of the IBDS maturation; (2) abnormal reaction patterns occur mainly in NH, whilst BD/PT < 0.5 in BA; and (3) lack of intrahepatic bile ducts in infants aged less than 1 year is an adverse prognostic factor independent from aetiology of neonatal liver disease.

Genetics of adenocarcinomas of the small intestine: frequent deletions at chromosome 18q and mutations of the SMAD4 gene.

The small intestinal mucosa makes up about 90% of the total surface of the gastrointestinal tract. However, adenocarcinomas arise rarely in this location. To elucidate genetic alterations underlying tumour development in the small intestine we investigated 17 sporadic adenocarcinomas. By comparative genomic hybridization recurrent gains of chromosomal material were found at chromosomes 7, 8, 13q, and 20 (5/17, each), while non-random losses were seen at 8p, 17p (4/17, each), and 18 (8/17 cases). Deletions at 5q, the location of the APC tumour suppressor gene, were seen in three cases. Microsatellite analysis with markers on chromosomal arms 1p, 5q, 8p, 17p, 18q, 19p, and 22q revealed a microsatellite instable phenotype in two cases and a high frequency of loss at 18q21-q22 (80%). Given the high incidence of 18q21-q22 deletions, we performed sequencing analysis of SMAD4, a downstream component of the TGFbeta-pathway, located at 18q21. Four tumours displayed mutations in highly conserved domains of the gene indicating disruption of TGFbeta-signalling. Our data reveal complex genetic alterations in sporadic small intestinal carcinomas. However, most tumours share deletions of 18q21-q22, which frequently target SMAD4. This indicates that disruption of TGFbeta-signalling plays a critical role in small intestinal tumorigenesis.

Chromosomal imbalances in sporadic neuroendocrine tumours of the thymus.

Neuroendocrine (carcinoid) tumours of the thymus are rare neoplasms characterized by a highly malignant clinical behavior. Some of these tumors are associated with MEN1. In this study we evaluated 10 cases of sporadic thymic neuroendocrine tumours using immunohistochemistry and comparative genomic hybridization (CGH). All tumours showed a diffuse expression of neuron specific enolase (NSE) and synaptophysin. Chromosomal imbalances were detected in 8/10 cases, the most frequent gains were seen on chromosome Xp (3/10 cases), 7p, 7q, 11q, 12q, and 20q (2/10 each), losses were most frequently detected at 6q (5/10 each), 6p (3/10 each), 4q (3/10 each), 3p, 10q, 11q and 13 q (2/10 each). These CGH data show a degree of overlap with chromosomal imbalances commonly observed in advanced thymomas.

Immunohistochemical and cytogenetic characterization of acantholytic squamous cell carcinoma of the breast.

Primary acantholytic squamous cell carcinoma (ASCC) of the breast is a rare and aggressive variant of invasive breast cancer. Here we report two new cases of ASCC and their immunohistochemical and cytogenetic characterization. One case was associated with systemic metastases and death and the other with local failure prior to loss of follow-up. Using comparative genomic hybridization (CGH), both tumors showed a high overall number of chromosomal imbalances with a similar pattern of gains and losses. Genetic aberrations common to both tumors included losses at 3p11-p25, 5q21-q31, 8p, 9, 13p13-q21, 16q12-q21, and 17p and gains at 1q31-qter, 7p, 18q12-qter, 19q, and 20. Immunohistochemically, the tumors were characterized by high proliferative activity, an uncommon cytokeratin expression profile, reduced E-cadherin staining, and overexpression of p53 and epithelial growth factor receptor (EGFR). The results of our analyses suggest that genetic alterations observed in ASCC of the breast include imbalances commonly observed in both mammary adenocarcinoma and squamous cell carcinoma of other locations. Furthermore, the overexpressed EGFR could be a possible therapeutic target for individual cases of this aggressive tumor type.

Oncogenetic tree models based on cytogenetic data: new insights into the development of epithelial tumors of the thymus.

Epithelial tumors of the thymus are rare neoplasms typically arising in the anterior mediastinum. There is an ongoing discussion whether thymomas of different histological subtypes form a biological continuum or represent distinct biological entities. To further investigate this question, we performed a statistical analysis of CGH data of 65 previously published cases. Losses of 3p, 6p, 6q, 13q, 16q, and 17p, as well as gains on 1q, were found in at least 10% of the cases. Comparing the data from B2, B3, and C thymomas, we noted an increasing complexity of karyotypes that may be well explained by a sequential order of these types. The frequencies of losses on 16q and 17p show a significant trend with respect to the sequence from B2 to B3 and C thymomas, indicating that these aberrations may be important events in the transition between these tumor types. To identify pathways of genetic development and progression of thymomas, we used oncogenetic tree models representing the dependencies between recurrent chromosomal aberrations. This analysis suggests that gains on the long arm of chromosome 1 occur early in tumor development and are correlated with losses on 6p and 6q. There is a weak correlation with losses on 16q and 17p, which appear to be late events. An independent pathway leads to losses on 3p and 13q, which are closely correlated. Our results indicate that the development of thymomas seems to be in some part a multistep mechanism. Oncogenetic tree models are a helpful means to determine developmental pathways of tumors arising from the same progenitor cell, as shown here for thymomas.

Cystic thymoma.

Thymic cysts are rare lesions located along the anatomical course of the third pharyngeal pouch. While most of the cases represent congenital cysts, they may also be related to neoplasms. We report a case of a micronodular thymoma with lymphoid stroma, which was completely built of small cysts, discuss the pathologic features of this tumor type and review the etiology and other aspects of thymic cysts.

Identification of a CTL4/Neu1 fusion transcript in a sialidosis patient.

The deficiency of the lysosomal neuraminidase (NEU1; sialidase) causes the storage disorder sialidosis with symptoms ranging from eye abnormalities and neurological disturbances to skeletal malformations, mental retardation and early death. Sialidosis patients encompassing a wide spectrum of clinical symptoms were screened for mutations in neu1. We identified the same homozygous interstitial deletion (11 kb) in two patients causing the fusion of exon 10 of CTL4 (New Gene 22; NG22) with the 3'-UTR of neu1. In one patient we found the resulting CTL4/Neu1 fusion transcript, in the other we detected an alternatively spliced CTL4 transcript (retention of intron 9).

Flow cytometric detection of spontaneous apoptosis in human breast cancer using the TUNEL-technique.

Microscopic detection of structural alterations is the most reliable method to identify apoptotic cells, which however, does not allow any correlation with cell cycle phases. Discrimination of individual cells within solid human tumors undergoing apoptotic death is possible by flow cytometry where apoptotic cells appear in a hypodiploid sub G0/1-peak as a consequence of partial DNA loss. To refer induction of apoptosis to cell cycle phases we adopted the terminal deoxynucleotidyl transferase nick-end-labelling (TUNEL) technique to flow cytometry which enables the detection of cellular DNA content and DNA fragmentation by multiparametric analysis. One thousand seven hundred human breast carcinomas were screened. In 40 cases (2.3%) of 1700 carcinomas we detected a hypodiploid sub -G0/1 apoptotic peak. The spontaneous apoptotic fractions within individual tumors ranged between 1.5 and 25%. A correlation (r(2)=0.78) was found between apoptotic cells in sub-G0/1-peak measured by DNA-cytometry and TUNEL positive cells measured by multiparametric cytometry, because TUNEL reaction signed also cells with strand breaks. High proliferation indices correspond well (r(2)=0.807) with the increased amount of TUNEL positive cells. Multiparametric flow cytometry for the combined determination of DNA-content and DNA-fragmentation by TUNEL offers not only the advantage of a higher apoptosis sensitivity but also enables the quantification of DNA fragmentation related to any cell cycle phase.

Expression of cell cycle-related gene products in different forms of primary versus recurrent PVNS.

Expression patterns of cell cycle regulating gene products and Ki-67 in proliferating synovial cells of primary and recurrent pigmented villonodular synovitis (PVNS) in localized and diffuse lesions were examined by immunohistochemistry. Alterations of cell cycle-related proteins were seen in 98.7% of analyzed lesions. Both RB- and p53 pathways play a role in cell cycle dysregulation in PVNS. The RB pathway was more frequently altered in primary disease, while alterations of the p53 pathway seemed to be more important in recurrent lesions, regardless of the histomorphological type of disease. Ki-67 proliferation rate was elevated in recurrent tumors.

Wnt-signaling and apoptosis after neoadjuvant short-term radiotherapy for rectal cancer.

Recent surgical concepts for primary rectal cancer include the combination of surgery and short-term neoadjuvant radiotherapy (STNR). This is usually given in a dose of 25 Gy over five days in order to reduce local recurrence rates. Clinical studies have shown that local recurrence is found in some patients despite STNR. We identified molecular patterns of the Wnt- and apoptosis pathways as well as expression of junction-associated molecules in rectal cancer specimens of patients who received STNR and in those who did not. Expression patterns were examined by immunohistochemistry and molecular techniques such as LightCycler RT-PCR and Western blot analysis in 25 sporadic rectal adenocarcinoma specimens derived from STNR-patients or non-pretreated donors, respectively. The molecular pattern in response to STNR was heterogeneous and was reflected by responders who show activation of apoptosis and cellular remodeling, whereas the group of non-responders from STNR did not show such reaction and was very similar to untreated controls. Enhanced expression of beta-catenin was generally mediated by STNR, but exclusively in the responder group impaired expression of c-Myc and junction-associated molecules as well as cleavage of poly-ADP-ribose polymerase and of the caspase substrate cytokeratin 19 were found. The molecular profile suggests that STNR interferes with Wnt-signaling and c-Myc expression. STNR in its present form is not suitable to fully complete the sequence of apoptosis in all rectal adenocarcinomas.

Impaired expression of acyl-CoA-synthetase 5 in epithelial tumors of the small intestine.

Fatty acids are implicated in tumorigenesis, but data are limited concerning endogenous fatty acid metabolism of tumor cells in adenomas and adenocarcinomas of the small intestine. The recently cloned human acyl-CoA-synthetase 5 (ACS5) is predominantly found in the small intestine and represents a key enzyme in providing cytosolic acyl-CoA thioesters. Protein synthesis and mRNA expression of ACS5 were studied in human intestinal tissues using different methods, including a newly established monoclonal antibody. In the healthy small intestine, expression of ACS5 was restricted to the villus surface epithelium but was not detectable in enterocytes lining crypts. ACS5 protein and mRNA were progressively diminished in epithelial cells of adenomas and adenocarcinomas of the small intestine. In conclusion, altered expression of ACS5 is probably related to the adenoma-carcinoma sequence of small intestinal epithelial tumors due to an impaired acyl-CoA thioester synthesis.

Somatic mutations in familial adenomatous polyps. Nuclear translocation of beta-catenin requires more than biallelic APC inactivation.

Germline mutations of the APC gene cause familial adenomatous polyposis coli (FAP). APC inactivation results in dysregulation of wnt/wingless signaling and contributes to chromosomal instability in vitro. To investigate somatic alterations that follow a known germline mutation and contribute to the transition from normal to neoplastic mucosa, we studied 10 adenomatous polyps from a 27-year-old patient with an APC germline mutation at codon 554. Chromosomal imbalances were analyzed by comparative genomic hybridization; APC and K-ras were screened for somatic mutations. Before DNA analysis, the polyps were bisected to compare the genetic alterations with the corresponding immunohistologic phenotype of beta-catenin, a proto-oncogene product degraded by the APC tumor suppressor. Gains at chromosome 20 were the most frequent chromosomal alterations (6 polyps). Losses were found predominantly at chromosome 4q (3 polyps). A K-ras mutation was seen in 1 polyp, while all polyps displayed somatic intragenic APC mutations. Comparative immunohistologic analysis revealed strong membranous staining for beta-catenin in all adenomatous polyps, but only 1 adenoma showed nuclear accumulation. Our results suggest chromosomal aberrations contribute early to the progression of adenomatous polyps after biallelic APC inactivation. APC inactivation itself is insufficient for immunohistochemically detectable nuclear translocation of beta-catenin.

Oligoclonal T-cell populations in an inflammatory pseudotumor of the pancreas possibly related to autoimmune pancreatitis: an immunohistochemical and molecular analysis.

Inflammatory pseudotumors (IPT), also known as inflammatory myofibroblastic tumors (IMT), are benign inflammatory processes that may have an infectious etiology and are very rare in the pancreatico-biliary region. Recent studies suggest a biological distinction between IPT and IMT, the latter being a true neoplastic process. We describe a case of pancreatic IPT, originally diagnosed as malignancy, which presumably recurred 4 months after the operation. Histologically, the tumor consisted of a smooth muscle actin and CD68-positive spindle cell population and a more abundant mononuclear inflammatory cell population, primarily composed of macrophages and T-lymphocytes. Inflammatory cells were the source of connective tissue growth factor and transforming growth factor-beta1 and tended to accumulate around nerves and blood vessels, as well as around residual pancreatic parenchymal elements, where an intense angiogenetic response was detected. Comparative genomic hybridization analysis of the tumor showed no chromosomal imbalances. Polymerase chain reaction-based analysis of T-cell receptor gamma gene rearrangement revealed an oligoclonal pattern. These findings suggest that the pathogenesis of aggressive cases of IPT could be related to the development of an intense and self-maintaining immune response, with the emergence of clonal populations of T-lymphocytes. The relation of the pancreatic IPT to autoimmune pancreatitis is emphasized.

Microcystic tubulopapillary carcinoma of the pancreas: a new tumor entity?

An unusual pancreatic tumor with microcystic and tubulopapillary features was observed in a 53-year-old woman. The tumor presented as a large, focally cystic mass in the head of the pancreas, which compressed the surrounding structures. The histological and immunohistochemical analysis revealed a neoplasm that could not be assigned to any of the known pancreatic tumor types. At the molecular level, the tumor showed inactivation of the DPC4/SMAD4 gene, deletion of exon 1 of the p16(INK4A) gene and a point mutation at codon 34 (GGA>AGA) of beta-catenin. Transcriptional profiling analyses and subsequent correspondence cluster analysis demonstrated that the transcriptional profile of the tumor differed distinctly from that of ductal adenocarcinomas, pancreatic cystic tumors and normal pancreatic tissues. These data suggest that the neoplasm most likely represents a new pancreatic tumor entity, which we would like to refer to as microcystic tubulopapillary tumor.

Differential diagnosis of non-epithelial tumors of the pancreas: malignant non-epithelial pancreatic tumor with focal pigmentation.

Non-epithelial tumors only rarely affect the pancreas. In this report, we describe a malignant non-epithelial tumor with combined characteristics of malignant peripheral nerve sheath tumor (MPNST) and malignant melanoma. To more closely define the differential diagnosis of MPNST with focal pigmentation versus metastatic melanoma resembling MPNST, the tumor was investigated using histomorphology, immunohistochemistry, electron microscopy, and comparative genomic hybridization. As a result, from these analyses and from clinical findings, the diagnosis of a pancreatic MPNST with focal pigmentation was favored. However, the diagnosis of a malignant melanoma or a composite tumor could not be definitely ruled out, due to the considerable morphological and genotypical overlap between both entities, which can be explained by the close histogenetic relationship between both tumor entities.

Cytokine/chemokine messenger-RNA expression profiles in ulcerative colitis and Crohn's disease.

To define mediator profiles in inflamed and noninflamed areas in inflammatory bowel disease (IBD) we analyzed the expression of 35 messenger-RNAs (mRNAs) encoding cytokines, chemokines, and some related molecules in transmural gut tissues (n=138) from patients with ulcerative colitis (UC), Crohn's disease (CD), and inflammatory and normal controls by real-time quantitative reverse transcription polymerase chain reaction. Using sample collectives with a comparable degree of inflammation, most parameters investigated showed similarly increased mRNA expression levels in both active UC and CD. This included proinflammatory cytokines, but also interferon (IFN) gamma and several IFN-gamma inducible chemokines. Only macrophage inflammatory protein (MIP)-2alpha mRNA was expressed at higher levels in inflamed UC vs. CD. IH revealed that MIP-2alpha protein was produced mainly by intestinal epithelial cells. Importantly, in histologically noninflamed/inactive IBD samples mRNAs for several mediators were significantly enhanced, accompanied by elevated levels of migration-inhibition factor related protein (MRP) 14 transcripts. CD14 positive macrophages were found especially in noninflamed/inactive UC, many of which coexpressed the RFD-7 antigen. Our results indicate a substantial overlap in cytokine/chemokine mRNA expression in UC and CD. Elevated mediator expression is evident in noninflamed/inactive areas in both diseases. Local recruitment of MRP-14 positive leukocytes might contribute to this phenomenon. In inactive UC a phenotypically altered population of macrophages expressing CD14 might play an additional role.

The role of platelet derived growth factor in endomyocardial biopsies shortly after heart transplantation in relation to postoperative course.

OBJECTIVE: Platelet-derived growth factor (PDGF) plays an important role in structural alterations of blood vessels after heart transplantation (HTx). The aim of this study was to clarify the effect of peritransplant injury and postoperative complications on the expression of PDGF ligand and receptor. METHODS: Right ventricular endomyocardial biopsies were collected from 46 patients before implantation, and then 1 and 2 weeks after HTx. According to the clinical course in the first postoperative year and to histopathological evaluation (based on the standardised 'International Society for Heart and Lung Transplantation' grading system) three groups were formed: (a) clinical uneventful course; (b) histologically and/or serologically proven cardiac or systemic infections; and (c) acute rejection episodes > or =grade 3A. Both, infections and rejections were detectable after the second postoperative week. The expression of PDGF AA/BB and PDGF receptors alpha/beta was examined immunohistochemically. The infiltrating cells were characterised by using monoclonal antibodies against CD3, CD4, CD8, CD57 and CD68. RESULTS: Only endothelial cells revealed a relevant expression of PDGF ligand and receptor. Prior to implantation there was no or only weak reactivity of single cells for all PDGF factors. One week after HTx a significantly increased immunoreactivity of all PDGF factors was observed in all groups. Two weeks after HTx the expression of PDGF AA in the infection group and the expression of all four PDGF factors in the rejection group remained significantly elevated. In contrast, in the group with an uneventful course there was no statistical difference in the expression of all the four PDGF factors. Compared with the uneventful group, there were significantly more CD3+ cells in the infection and rejection group at all three time points. Two weeks after HTx, the rejection group showed a significantly elevated number of CD3+ cells compared to the values before implantation. Two weeks after HTx there were significant more CD68+ cells in the infection and rejection group compared with before implantation. CONCLUSIONS: One week after HTx the peritransplant injury predominantly influences the endothelial expression of PDGF ligand and receptor. In the first postoperative week, expression of PDGF could be detected. The persistence of evaluated PDGF expression might be of prognostic value in terms of a risk for either infection or rejection. These patients should be carefully monitored.

Loss of SMAD4 function in small intestinal adenocarcinomas: comparison of genetic and immunohistochemical findings.

Despite morphological similarities between adenocarcinomas of the small and the large intestine, recent evidence suggests that both tumor types follow different genetic pathways. In particular, inactivation of the APC tumor suppressor gene, a characteristic alteration of colorectal carcinomas, does not seem to play a significant role in sporadic small intestinal tumorigenesis. We could recently show that inactivating mutations of the SMAD4 gene frequently occur in small intestinal adenocarcinomas. To further elucidate the role of SMAD4 dysfunction for tumor development in the small intestine, we immunohistochemically analyzed 20 sporadic, non-ampullary carcinomas for the expression of the SMAD4 protein. We further determined homozygous SMAD4 gene deletions by real time PCR and compared SMAD4 immunohistochemical data with SMAD4 genetic data. Immunohistochemistry was negative for the tumor cells in two (10%) cases and strongly reduced in four (20%). Negative immunohistochemical staining corresponded with homozygous gene deletions. A regular or only slightly reduced staining pattern was noted in 14 carcinomas, including four tumors with previously identified SMAD4 missense and frame shift mutations. In conclusion, our data suggest a significant role of impaired SMAD4 function in the pathogenesis of small intestinal adenocarcinomas. Furthermore, our results show that SMAD4 immunohistochemistry may serve as a surrogate for analysis of homozygous gene deletions. However, the method fails to identify SMAD4 inactivation due to missense mutations.

Single nucleotide insertion in the 5'-untranslated region of hepatitis C virus with clearance of the viral RNA in a liver transplant recipient during acute hepatitis B virus superinfection.

Hepatitis C virus (HCV) infection is an important etiology in patients undergoing orthotopic liver transplantation (OLT) world-wide. Antiviral therapy-related clearance of HCV RNA may occur both in patients with chronic HCV infection and in transplanted patients for HCV-related liver cirrhosis, but the role of the 5'-untranslated region (UTR) of HCV containing the internal ribosome entry site (IRES), which directs the translation of the viral open reading frame has not hitherto been evaluated. We studied the 5'-UTR in an HCV-infected recipient of a liver graft that showed spontaneous clearance of HCV RNA during an acute hepatitis B virus (HBV) superinfection. Sequencing of the 5'-UTR of HCV showed a nucleotide A insertion at position 193 of the IRES.