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Background & Aims: Hepatocellular adenomas (HCA) are benign liver tumors mainly related to oral contraception and classified into 4 molecular subgroups: inflammatory (IHCA), HNF1A-inactivated (H-HCA), β-catenin-activated (bHCA) or unclassified (UHCA). Glycogen storage disease type I (GSD) is a rare hereditary metabolic disease that predisposes to HCA development. The aim of our study was to characterize the molecular profile of GSD-associated HCA., Methods: We characterized a series of 25 HCAs developed in 15 patients with GSD by gene expression and DNA sequence of HNF1A, CTNNB1, IL6ST, GNAS, and STAT3 genes. Moreover, we searched for glycolysis, gluconeogenesis, and fatty acid synthesis alterations in GSD non-tumor livers and compared our results to those observed in a series of sporadic H-HCA and various non-GSD liver samples., Results: GSD adenomas were classified as IHCA (52%) mutated for IL6ST or GNAS, bHCA (28%) or UHCA (20%). In contrast, no HNF1A inactivation was observed, showing a different molecular subtype distribution in GSD-associated HCA from that observed in sporadic HCA (p = 0.0008). In non-tumor GSD liver samples, we identified glycolysis and fatty acid synthesis activation with gluconeogenesis repression. Interestingly, this gene expression profile was similar to that observed in sporadic H-HCA., Conclusions: Our study showed a particular molecular profile in GSD-related HCA characterized by a lack of HNF1A inactivation. This exclusion could be explained by similar metabolic defects observed with HNF1A inactivation and glucose-6-phosphatase deficiency. Inversely, the high frequency of β-catenin mutations could be related to the increased frequency of malignant transformation in hepatocellular carcinoma., (Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Somatic mutations activating telomerase reverse-trancriptase promoter were recently identified in several tumour types. Here we identify frequent similar mutations in human hepatocellular carcinomas (59%), cirrhotic preneoplastic macronodules (25%) and hepatocellular adenomas with malignant transformation in hepatocellular carcinomas (44%). In hepatocellular tumours, telomerase reverse-transcripase- and CTNNB1-activating mutations are significantly associated. Moreover, preliminary data suggest that telomerase reverse-trancriptase promoter mutations can increase the expression of telomerase transcript. In conclusion, telomerase reverse-trancriptase promoter mutation is the earliest recurrent genetic event identified in cirrhotic preneoplastic lesions so far and is also the most frequent genetic alteration in hepatocellular carcinomas, arising from both the cirrhotic or non-cirrhotic liver.

Hepatocellular adenoma usually arises in the absence of significant fibrosis. Herein, we report seven patients with serum amyloid A-positive hepatocellular neoplasm, which shares features with inflammatory hepatocellular adenoma arising in alcoholic cirrhosis. Seven patients (two women and five men, age range 41-67 years) with hypervascular hepatocellular nodules associated with alcoholic cirrhosis were retrieved from our pathological files (1997-2011). The hepatocellular nodules were multiple (>3) in all patients and 17 nodules were histologically examined. We surveyed the immunoreactivity for serum amyloid A, glutamine synthetase, and glypican-3 in the hepatocellular nodules and control lesions, including 5 focal nodular hyperplasia, 18 dysplastic nodules, and 54 hepatocellular carcinomas in various background diseases. In all, 15 of 17 nodules showed strong and distinct immunoreactivity for serum amyloid A, sharing features with inflammatory hepatocellular adenoma. The serum amyloid A-positive hepatocellular neoplasms showed increased cellular density, inflammatory infiltrate, sinusoidal dilatation, and ductular reaction to various degrees. Although about a half of dysplastic nodules and hepatocellular carcinomas showed focal immunoreactivity for serum amyloid A, the extent of serum amyloid A expression was significantly higher in serum amyloid A-positive hepatocellular neoplasms, than in control nodules. The serum amyloid A-positive hepatocellular neoplasms did not show the overexpression of glutamine synthetase or immunoreactivity for glypican-3. In contrast, most hepatocellular carcinomas showed the overexpression of glutamine synthetase and immunoreactivity for glypican-3, irrespective of background diseases. In conclusion, this study highlights a characteristic group of hepatocellular neoplasms arising in alcoholic cirrhosis, which share features with inflammatory hepatocellular adenomas. These serum amyloid A-positive hepatocellular neoplasms may be a new type of inflammatory hepatocellular tumors in alcoholic patients.

Phenotypic identification of focal nodular hyperplasia (FNH) and hepatocellular adenoma (HCA) subtypes using immunohistochemical markers has been developed from their molecular characteristics. Our objective was to evaluate the sensitivity of these markers in the definitive diagnosis of these lesions by core needle biopsies. A total of 239 needle biopsies paired with their surgical resection specimen (group A) or without an associated resection specimen (group B) were reviewed. Using a step-by-step algorithm after standard staining, appropriate immunostaining analyses were performed to determine the certainty of diagnosis of FNH, HNF1α-inactivated HCA, inflammatory HCA, β-catenin-activated HCA, or unclassified HCA. The diagnosis of FNH was certain or probable on routine stains in 53% of needle biopsies of group A, whereas after glutamine synthetase staining, the diagnosis was certain in 86.7% as compared with 100% on the corresponding surgical specimen (P=0.04). In needle biopsies of group A, the diagnosis of HCA was certain on routine stains in 58.6% as compared with 94.3% on surgical specimens. After specific immunostaining, diagnosis was established on biopsies with 74.3% certainty, including all HCA subtypes, with similar distribution in surgical specimens. For each "certain diagnosis" paired diagnostic test (biopsy and surgical specimen), a positive correlation was observed (P<0.001). No significant difference was observed between groups A and B for FNH (P=0.714) or for HCA subtypes (P=0.750). Compared with surgical specimens, immunohistochemical analysis performed on biopsies allowed the discrimination of FNH from HCA and the identification of HCA subtypes with good performance.

Objective: Hepatocellular adenomas (HCAs) are divided into three subtypes according to genotype and phenotype. The two main subgroups are hepatocyte nuclear factor 1α (HNF1α)-inactivated HCA and inflammatory HCA. Specific imaging features of these subgroups of adenoma have been delineated with MRI. The aim of this study was to document the contrast-enhanced sonographic (CEUS) features specific for adenoma subtypes and to correlate them with MRI findings., Materials and Methods: We retrospectively analyzed data on 38 patients with HCA confirmed at pathologic examination in all cases. All cases were classified with MRI., Results: HNF1α-inactivated HCA (n = 16) was found to have a homogeneous hyperechoic aspect at baseline gray-scale sonography, isovascularity or moderate hypervascularity with mixed filling in the arterial phase, and isoechogenicity in the portal and late portal venous phases. Homogeneous hyperechogenicity during B-mode sonography was the most specific pattern (sensitivity, 88%; specificity, 91%) and correlated with diffuse fat repartition observed on MR images obtained with chemical-shift sequences. In inflammatory HCA (n = 17) the association of arterial hypervascularity with centripetal filling, linear vascularities, peripheral rim of sustained enhancement, and central washout in the late venous phase was specific (sensitivity, 64%; specificity, 100%). Discrepancy between delayed washout during CEUS and sustained enhancement during MRI could be related to differences between gadolinium and microbubbles in diffusing in the interstitial spaces. In the five other HCA cases (four unclassified, one β-catenin activated) CEUS showed characteristics of benign hepatocellular tumors with no specific features., Conclusion: HNF1α-inactivated HCA and inflammatory HCA had characteristic CEUS patterns. Delayed washout, an unusual finding in benign hepatic lesions, is of particular interest and was a characteristic of inflammatory HCA subtype.

Background: Autoimmune hyperlipidemia (AIH) is a rare cause of secondary hyperlipidemia. A few cases of AIH have been reported in multiple myeloma., Material and Methods: A female in her fifties was referred to the outpatient clinic presenting with headache, blurred vision and skin rash. Physical examination with subsequent laboratory and histological examinations revealed severe hyperlipidemia secondary to secretory multiple myeloma with monoclonal IgG kappa protein and erythrocytosis secondary to a erythropoietin secreting adenoma in the liver., Results and Interpretation: Treatment for multiple myeloma (induction treatment and autologous hematological stem cell transplantation) gained partial remission and was associated with normalization of serum lipids. There was no need for further medical treatment of the hyperlipidemia. Three years after the initial treatment, serum concentrations of triglycerides and total cholesterol increased in parallel with monoclonal IgG kappa protein. Total cholesterol and triglycerides decreased and remained within the reference ranges after retreatment with a second autologous stem cell transplantation. Surgical removal of the hepatic adenoma caused normalisation of the erythropoietin concentration and resolution of the erythrocytosis. The present case reports two rare complications (AIH and erythrocytosis) to multiple myeloma and hepatic adenoma, with regression of complaints and normalisation of laboratory tests after adequate treatment of underlying diseases.

Ligand-independent constitutively active gp130 mutants were described to be responsible for the development of inflammatory hepatocellular adenomas (IHCAs). These variants had gain-of-function somatic mutations within the extracellular domain 2 (D2) of the gp130 receptor chain. Cytokine-dependent Ba/F3 cells were transduced with the constitutively active variant of gp130 featuring a deletion in the domain 2 from Tyr-186 to Tyr-190 (gp130ΔYY). These cells showed constitutive phosphorylation of signal transducer and activator of transcription-3 (STAT3) and cytokine-independent proliferation. Deletion of the Ig-like domain 1 (D1) of gp130, but not anti-gp130 mAbs directed against D1, abolished constitutive activation of gp130ΔYY, highlighting that this domain is involved in ligand-independent activation of gp130ΔYY. Moreover, soluble variants of gp130 were not able to inhibit the constitutive activation of gp130ΔYY. However, the inhibition of constitutive activation of gp130ΔYY was achieved by the anti-gp130 mAb B-P4, which specifically inhibits gp130 signaling by IL-11 but not by other IL-6 type cytokines. IL-11 but not IL-6 levels were found previously to be up-regulated in IHCAs, suggesting that mutations in gp130 are leading to IL-11-like signaling. The mAb B-P4 might be a valuable tool to inhibit the constitutive activation of naturally occurring gp130 mutants in IHCAs and rare cases of gp130-associated hepatocellular carcinoma.

The present study investigated the potential role for activation of PPARα and CAR/PXR by potassium PFOS (K⁺ PFOS) with respect to the etiology of hepatic hypertrophy and hepatocellular adenoma in rats. Male Sprague-Dawley rats were fed K⁺ PFOS (20 or 100 ppm) for either 1, 7, or 28 days. Wyeth 14,643 (Wy 14,643, 50 ppm) and phenobarbital (PB, 500 ppm) were the controls for PPARα and CAR/PXR activation, respectively. Measurements included: plasma ALT, AST, cholesterol, triglycerides, and glucose; liver protein and DNA content; liver activities of palmitoyl CoA oxidase (ACOX), Cyp4A, CYP2B, and CYP3A; induction of liver CYP4A1, CYP2E1, CYP2B1/2, and CYP3A1 proteins (SDS-PAGE and Western blots); liver and thyroid microscopic histopathology, apoptotic index, and cell proliferation index. Terminal body weight was decreased by K⁺ PFOS (100 ppm) and Wy 14,643. All test-compound treatments increased liver weight. Plasma lipids were decreased by both PFOS and Wy 14,643. After treatment for 1 day, K⁺ PFOS (100 ppm), PB, and Wy 14,643 increased mean hepatic DNA concentration and total hepatic DNA, and total DNA remained elevated after treatment for 7 days and 28 days (PB and Wy 14,643 only). Hepatic P450 concentration was elevated after 7 and 28 days by K⁺ PFOS and by PB. K⁺ PFOS and Wy 14,643 increased liver activities of ACOX and CYP4A as well as increased liver CYP4A1 protein. By 28 days of treatment, K⁺ PFOS and PB increased liver activities of CYP2B and CYP3A as well as increased liver CYP2B1/2 and CYP3A1 proteins, and Wy 14,643 increased CYP2B enzyme activity to a slight extent. All test compounds increased the liver cell proliferative index and decreased the liver apoptotic index. No histological changes of the thyroid were noted; however, PB and WY increased thyroid follicular cell proliferation index (seven-day treatment only), while K⁺ PFOS did not. The thyroid follicular cell apoptotic index did not differ between groups. The hepatomegaly and hepatocellular adenoma observed after dietary exposure of Sprague-Dawley rats to K⁺ PFOS likely are due to the increased expression of xenosensor nuclear receptors PPARα and CAR/PXR. Given the markedly lower or absent response of human hepatocytes to the proliferative stimulus from activation of PPARα and CAR/PXR, the hepatocellular proliferative response from activation of these receptors by PFOS observed in rats is not expected to be of human relevance., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Background & Aims: Mosaic G-protein alpha-subunit (GNAS)-activating mutations are responsible for the McCune-Albright (MCA) syndrome. This oncogene that activates the adenylate cyclase is also mutated in various tumor types leading to the accumulation of cyclic-AMP. Identification of a hepatocellular adenoma (HCA) in two MCA patients led us to search for GNAS activation in benign and malignant hepatocellular carcinogenesis., Methods: GNAS mutations were screened by sequencing 164 HCA, 245 hepatocellular carcinoma (HCC), and 17 fibrolamellar carcinomas. Tumors were characterized by quantitative RT-PCR, gene mutation screening and pathological reviewing. The consequences of wild type and mutant GNAS expression were analyzed in hepatocellular cell lines., Results: A somatic GNAS-activating mutation was identified in 5 benign tumors and in 2 HCC. In benign tumors, GNAS mutations were exclusive from HNF1A, CTNNB1, and IL6ST mutations whereas one HCC demonstrated both CTNNB1 and GNAS mutations. Quantitative RT-PCR showed an activation of the IL-6 and interferon pathways in GNAS-mutated tumor tissues. Accordingly, pathological reviewing identified in GNAS-mutated tumors an inflammatory phenotype characterized by fibrosis and STAT3 activation. We further demonstrated in HCC cell lines that GNAS mutant expression induced inflammatory response and STAT3 activation., Conclusions: We identified for the first time the association between two rare diseases, MCA syndrome and HCA occurrence, but also that somatic GNAS-activating mutations in sporadic benign and malignant liver tumors are characterized by an inflammatory phenotype. These results showed a cross-talk between cyclic-AMP and JAK/STAT pathways in liver tumors and they reinforce the role of STAT3 activation in liver tumorigenesis., (Copyright © 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Aims: Patients with hepatocellular carcinoma (HCC) usually present with advanced disease and rarely qualify for curative therapy. Immunohistochemical markers that help to discriminate benign from malignant processes early, and that have prognostic significance, would be useful. Expression of the oncofetal protein insulin-like growth factor II mRNA-binding protein 3 (IMP3) in malignant cells of different tumour types correlates with reduced overall survival., Methods and Results: Tissue microarrays (TMAs) containing 55 normal liver samples, 365 HCCs (122 with corresponding non-tumorous liver), 10 hepatocellular adenomas, 13 focal nodular hyperplasias and nine dysplastic nodules from western European patients were stained for IMP3. IMP3 was analysed in 61 core needle biopsies and findings were compared to glypican-3 and CD34. HCCs in TMAs were strongly positive for IMP3 in 18.4% of cases compared to absent expression in normal and non-tumorous liver tissue and benign liver tumours. Patients with IMP3 expression in HCCs showed significantly poorer overall survival in multivariate analysis (P = 0.044). Of the 61 core needle biopsies analysed, 32 (52.5%) of the HCCs were IMP3-positive., Conclusions: In core needle biopsies, IMP3 expression seems to be of limited use as a single marker for the diagnosis of HCC, given a sensitivity of 52%, but it may be helpful in combination with other markers., (© 2011 Blackwell Publishing Limited.)

Propiconazole induces hepatocellular carcinomas and hepatocellular adenomas in mice and promotes liver tumors in rats. Transcriptional, proteomic, metabolomic and biochemical studies of hepatic tissues from mice treated with propiconazole under the conditions of the chronic bioassay indicated that propiconazole induced oxidative stress. Here we sought to identify the source of the reactive oxygen species (ROS) induced by propiconazole using both AML12 immortalized mouse hepatocytes in culture and liver tissues from mice. We also sought to further characterize the nature and effects of ROS formation induced by propiconazole treatment in mouse liver. ROS was induced in AML12 cells by propiconazole as measured by fluorescence detection and its formation was ameliorated by N-acetylcysteine. Propiconazole induced glutathione-S-transferase (GSTα) protein levels and increased the levels of thiobarbituric acid reactive substances (TBARS) in AML12 cells. The TBARS levels were decreased by diphenylene iodonium chloride (DPIC), a cytochrome P450 (CYP) reductase inhibitor revealing the role of CYPs in ROS generation. It has been previously reported that Cyp2b and Cyp3a proteins were induced in mouse liver by propiconazole and that Cyp2b and Cyp3a proteins undergo uncoupling of their CYP catalytic cycle releasing ROS. Therefore, salicylic acid hydroxylation was used as probe for ROS formation using microsomes from mice treated with propiconazole. These studies showed that levels of 2,3-dihydroxybenzoic acid (an ROS derived metabolite) were decreased by ketoconazole, melatonin and DPIC. In vivo, propiconazole increased hepatic malondialdehyde levels and GSTα protein levels and had no effect on hepatic catalase or superoxide dismutase activities. Based on these observations we conclude that propiconazole induces ROS in mouse liver by increasing CYP protein levels leading to increased ROS levels. Our data also suggest that propiconazole induces the hydroxyl radical as a major ROS form., (Published by Elsevier Ireland Ltd.)

Background: Liver carcinogenesis is associated with multiple genetic changes in affected cells, including alterations in the Hras signalling pathway., Aim: To define the biological contributions of Hras to mouse hepatocarcinogenesis, we quantified in vivo interactions between mutant Hras and other genetic alterations frequently associated with liver cancer, including overexpression of the transcription factor c-myc and the epidermal growth factor receptor ligand transforming growth factor alpha (TGFα)., Methods: To accomplish this aim, we initiated expression of an activated Hras in hepatocytes of adult mice with or without simultaneous overexpression of either c-myc or TGFα. Potential interactions also were assessed through the use of the comparative hepatocyte growth assay, a hepatocyte transplantation assay that measures effects of altered gene expression on hepatocyte growth in vivo., Results: Hras expression caused diffuse liver enlargement (hepatomegaly), and this phenotype was not changed by coexpression of c-myc or TGFα. Using the transplant system, we found that expression of mutant Hras alone was sufficient to induce hepatocyte focus growth in a quiescent liver. Paradoxically, adding expression of TGFα or c-myc reversed this Hras effect. Finally, the frequencies of transplant foci with the preneoplastic feature of extreme growth potential and of liver neoplasms were increased for Hras and both combinations when compared with control hepatocytes, but did not differ among oncogene-expressing groups., Conclusions: Hras-associated hepatocyte growth deregulation is not complemented by activation of c-myc or TGFα growth signalling pathways in mouse liver. This finding emphasizes the tissue-specific character of molecular growth regulation., (© 2011 John Wiley & Sons A/S.)

Objective: To characterize the natural history and factors related to hepatocellular adenoma (HCA) development in glycogen storage disease type Ia (GSD Ia)., Study Design: Retrospective chart review was performed for 117 patients with GSD Ia. Kaplan-Meier analysis of HCA progression among two groups of patients with GSD Ia (5-year mean triglyceride concentration ≤ 500 mg/dL and >500 mg/dL); analysis of serum triglyceride concentration, body mass index SDS, and height SDS between cases at time of HCA diagnosis and age- and sex-matched control subjects., Results: Logrank analysis of Kaplan-Meier survival curve demonstrated a significant difference in progression to HCA between the 5-year mean triglyceride groups (P = .008). No significant difference was detected in progression to adenoma event between sexes. Serum triglyceride concentration was significantly different at time of diagnosis of adenoma (737 ± 422 mg/dL) compared with control subjects (335 ± 195 mg/dL) (P = .009). Differences in height SDS (P = .051) and body mass index SDS (P = .066) approached significance in our case-control analysis., Conclusion: Metabolic control may be related to HCA formation in patients with GSD Ia. Optimizing metabolic control remains critical, and further studies are warranted to understand the pathogenesis of adenoma development., (Copyright © 2011 Mosby, Inc. All rights reserved.)

Inflammatory hepatocellular adenomas (IHCAs) are benign liver tumors. 60% of these tumors have IL-6 signal transducer (IL6ST; gp130) mutations that activate interleukin 6 (IL-6) signaling. Here, we report that 12% of IHCA subsets lacking IL6ST mutations harbor somatic signal transducer and activator of transcription 3 (STAT3) mutations (6/49). Most of these mutations are amino acid substitutions in the SH2 domain that directs STAT3 dimerization. In contrast to wild-type STAT3, IHCA STAT3 mutants constitutively activated the IL-6 signaling pathway independent of ligand in hepatocellular cells. Indeed, the IHCA STAT3 Y640 mutant homodimerized independent of IL-6 and was hypersensitive to IL-6 stimulation. This was associated with phosphorylation of tyrosine 705, a residue required for IL-6-induced STAT3 activation. Silencing or inhibiting the tyrosine kinases JAK1 or Src, which phosphorylate STAT3, impaired constitutive activity of IHCA STAT3 mutants in hepatocellular cells. Thus, we identified for the first time somatic STAT3 mutations in human tumors, revealing a new mechanism of recurrent STAT3 activation and underscoring the role of the IL-6-STAT3 pathway in benign hepatocellular tumorigenesis.

Background & Aims: A molecular and pathological classification system for hepatocellular adenomas (HCA) was recently introduced and four major subgroups were identified. We aimed to validate this adenoma classification system and to determine the clinical relevance of the subtypes for surgical management., Methods: Paraffin fixed liver tissue slides and resection specimens of patients radiologically diagnosed as HCA were retrieved from the department of pathology. Immunostainings included liver-fatty acid binding protein (L-FABP), serum amyloid A (SAA), C-reactive protein (CRP), glutamine synthetase (GS) and β-catenin., Results: From 2000 to 2010, 58 cases (71 lesions) were surgically resected. Fourteen lesions were diagnosed as focal nodular hyperplasia with a characteristic map-like staining pattern of GS. Inflammatory HCA expressing CRP and SAA was documented in 36 of 57 adenomas (63%). Three of these inflammatory adenomas were also β-catenin positive as well as GS positive and only one was CRP and SAA and GS positive. We identified eleven L-FABP-negative HCA (19%) and four β-catenin positive HCA (7%), without expression of CRP and SAA and with normal L-FABP staining, one of which was also GS positive. Six HCA were unclassifiable (11%). In three patients multiple adenomas of different subtypes were found., Conclusions: Morphology and additional immunohistochemical markers can discriminate between different types of HCA in>90% of cases and this classification, including the identification of β-catenin positive adenomas may have important implications in the decision for surveillance or treatment. Interpretation of nuclear staining for β-catenin can be difficult due to uneven staining distribution or focal nuclear staining and additional molecular biology may be required., (Copyright © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Telangiectatic hepatocellular adenoma is a rare, recently recognized subtype of hepatocellular adenoma that is often underrecognized by pathologists. We report a case of hepatocellular carcinoma arising within a pigmented telangiectatic hepatocellular adenoma in a noncirrhotic man with diffuse glutamine synthetase and nuclear β-catenin positivity. This case highlights malignant transformation of telangiectatic adenomas, and describes a previously unreported association between pigment deposition and telangiectatic adenoma. Radiology, gross pathology, and histopathology are shown. Review of the literature with attention to β-catenin and glutamine synthetase staining, malignant transformation, patient characteristics, the presence of Dubin-Johnson-like pigment, and genetic characteristics of telangiectatic adenomas are discussed.

Autophagy is a major pathway for degradation of cytoplasmic proteins and organelles, and has been implicated in tumor suppression. Here, we report that mice with systemic mosaic deletion of Atg5 and liver-specific Atg7⁻/⁻ mice develop benign liver adenomas. These tumor cells originate autophagy-deficient hepatocytes and show mitochondrial swelling, p62 accumulation, and oxidative stress and genomic damage responses. The size of the Atg7⁻/⁻ liver tumors is reduced by simultaneous deletion of p62. These results suggest that autophagy is important for the suppression of spontaneous tumorigenesis through a cell-intrinsic mechanism, particularly in the liver, and that p62 accumulation contributes to tumor progression.

The aim of this pilot study was to evaluate the use of PET/CT with (18)F-fluorocholine in the differentiation of hepatocellular adenoma (HCA) from focal nodular hyperplasia (FNH). Patients with liver lesions larger than 2 cm suspicious for HCA or FNH were prospectively included. All patients underwent PET/CT with (18)F-fluorocholine and histopathological diagnosis was obtained by either liver biopsy or surgery. The ratios between the maximum standardized uptake value (SUV) of the lesion and the mean SUV of normal liver parenchyma were calculated and a receiver operating characteristic (ROC) curve analysis was performed. Ten patients with FNH and 11 with HCA were included. The mean SUV ratio was 1.68±0.29 (±SD) for FNH and 0.88±0.18 for HCA (p<0.001). An SUV ratio cut-off value between 1.12 and 1.22 differentiated patients with FNH from those with HCA with 100% sensitivity and 100% specificity. This pilot study showed that PET/CT with (18)F-fluorocholine can differentiate HCA from FNH.

The histologic distinction between reactive processes and malignant neoplasms and between low-grade and high-grade tumors is not always straightforward and is sometimes extremely challenging. This is especially the case when the diagnostic material is a small biopsy specimen or a cytology specimen with scant cellularity. In addition, suboptimal processing and crush artifact may limit accurate diagnosis. A reliable diagnostic biomarker that preferentially highlights malignant processes and high-grade tumors would be very valuable in segregating these entities from reactive processes and low-grade lesions. Recent extensive studies have shown that an oncoprotein, insulin like-growth factor II messenger RNA binding protein-3, is not only a prognostic biomarker but also a diagnostic molecule. This review focuses on discussing the value of insulin like-growth factor II messenger RNA binding protein-3 in diagnostic pathology, with a focus on utilization of insulin like-growth factor II messenger RNA binding protein-3 in the discrimination of benign effusions from malignant effusions, malignant mesothelioma from mesothelial hyperplasia, carcinoids from high-grade neuroendocrine carcinomas, low-grade dysplasia from high-grade dysplasia, hepatocellular carcinoma from hepatic adenoma, cholangiocarcinoma and metastatic pancreatic ductal carcinoma from benign bile duct lesions, melanoma from nevi, and follicular thyroid carcinoma from follicular adenoma of the thyroid, as well as examining insulin like-growth factor II messenger RNA binding protein-3 expression in lymphomas of germinal center origin., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This study used tissue samples from male B6C3F1 mice treated with ethanol in drinking water (0%, 2.5%, or 5%) for 4 or 104 weeks. We tested whether chronic alcohol drinking promotes oxidative stress in the liver and characterized the mutation profile of spontaneous and ethanol-induced tumors. We show that ethanol does not cause detectable oxidative stress in the liver at any time point and acts by promoting H-ras mutated cells., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

The present study was performed to characterize immunohistochemically the expression levels of molecules related to not only xenobiotic and antioxidant functions but also cell proliferation and apoptosis in neoplastic lesions induced by the benzimidazole anthelmintic, oxfendazole (OX), at the late stage of its tumor promotion in a rat hepatocarcinogenesis model. Male F344 rats were initiated with an intraperitoneal injection of 200 mg/kg N-diethylnitrosamine, and 2 weeks later they were fed a diet containing 0% (basal diet) or 0.05% OX for 26 weeks. All animals were subjected to a two-thirds partial hepatectomy at week 3 and killed at week 28. Histopathologically, OX increased the incidence and multiplicity of altered foci (4.0- and 3.6-fold, respectively) and hepatocellular adenomas (HCAs) (3.0- and 5.5-fold, respectively). OX treatment induced 5.2- and 5.6-fold increases in the number of proliferating cell nuclear antigen (PCNA)-positive cells and single-stranded DNA (ssDNA)-positive cells in HCAs compared with the surrounding tissue, respectively. Staining for the cell cycle regulators P21 and C/EBPα and the AhR-regulated CYP1A1 molecules decreased but increased reactivity of the Nrf2-regulated, detoxifing/antioxidant molecules aldo-keto reductase 7 (AKR7) and glutathione peroxidase 2 (GPX2) were also seen in HCAs compared with the surrounding hepatocytes. These results suggest that dysregulation of cell proliferation and apoptosis and escape from oxidative stress elicited by OX treatment play an important role in OX-induced hepatocarcinogenesis in rats.

Objective: To study the expression and significance of GPC3, CD10 and CD34 in hepatocellular carcinoma (HCC), dysplastic nodules (DN), cirrhotic regenerative nodules (CRN), focal nodular hyperplasia (FNH) and hepatocellular adenoma (HA)., Methods: Immunohistochemical study for GPC3, CD10, CD34 and AFP was performed on 80 cases of HCC (30 cases of well-differentiated HCC and 50 cases of advanced HCC), 30 cases of DN (18 cases of high-grade DN and 12 cases of low-grade DN), 36 cases of CRN, 20 cases of FNH and 20 cases of HA., Results: (1) The positive expression rate of GPC3 was 92% (46/50) in advanced HCC, 66.7% (20/30) in well-differentiated HCC, 2/18 in high-grade DN, and 0 in low-grade DN, CRN, FNH and HA. The expression rate of GPC3 in well-differentiated HCC was lower than that in advanced HCC and higher than that in high-grade DN (P < 0.05). (2) The negative expression rate of CD10 was 78% (39/50) in advanced HCC, 43.3% (13/30) in well-differentiated HCC, 20% (4/20 and 4/20) in both FNH and HA, 2.8% (1/36) in CRN and 0 in both high-grade DN and low-grade DN. The occurrence of CD10-strongly positive cells was 2% (1/50) in advanced HCC, 16.7% (5/30) in well-differentiated HCC, 15/18 in high-grade DN, 11/12 in low-grade DN, 80.6% (29/36) in CRN and 60% (12/20 and 12/20) in both FNH and HA. The positive expression rate of CD10 in well-differentiated HCC was higher than that in advanced HCC and lower than that in high-grade DN, low-grade DN, CRN, FNH and HA (P < 0.05). (3) The positive expression rates of CD34 in advanced HCC and well-differentiated HCC ranged from 25% to 100% [and strongly positive in 76% (38/50) and 70% (21/30), respectively]. The rates in high-grade DN and low-grade DN ranged from 5% to 25% (and weakly positive in 16/18 and 10/12, respectively). In CRN, the rate ranged from 0 to 5% [and weakly positive in 27.8% (10/36)]. In FNH and HA, the positive rates ranged from 25% to 50%. The positive expression rate of CD34 in well-differentiated HCC was significantly higher than that in high-grade DN, low-grade DN, CRN, FNH and HA (P < 0.05). (4) The positive expression rate of AFP was 44% (22/50) in advanced HCC, 20% (6/30) in well-differentiated HCC, no expression in DN, LCN, LCN, FNH and HA. The positive expression rate of AFP in well-differentiated HCC was lower than that in advanced HCC and higher than that in LCN, FNH and HA. The different expression had statistical significance (P < 0.05)., Conclusions: GPC3 is a relatively sensitive and specific marker in pathologic diagnosis of HCC. When coupled with immunohistochemical results of CD34, CD10 and AFP, GPC3 is useful in differentiating HCC from DN, LCN, FNH and HA.

A retrovirus homologue gene of cellular cyclin D₁, walleye dermal sarcoma virus rv-cyclin gene (orf A or rv-cyclin), was expressed in the livers of zebrafish under the control of liver fatty acid-binding protein (lfabp) promoter. To prevent possible fatality caused by overexpression of the oncogene, the GAL4/upstream activation sequence (GAL4/UAS) system was used to maintain the transgenic lines. Thus, both GAL4-activator [Tg(lfabp:GAL4)] and UAS-effector [Tg(UAS:rvcyclin)] lines were generated, and the rv-cyclin gene was activated in the liver after crossing these two lines. Since no obvious neoplasia phenotypes were observed in the double-transgenic line, cancer susceptibility of the transgenic fish expressing rv-cyclin was tested by carcinogen treatment. Unexpectedly, transgenic fish expressing rv-cyclin gene (rvcyclin+) were more resistant to the carcinogen than siblings not expressing this gene (rvcyclin-). Lower incidences of multiple and malignant liver tumors were observed in rvcyclin+ than in rvcyclin- fish, and the liver tumors in the rvcyclin+ group appeared later and were less malignant. These results suggest that expression of rv-cyclin protects the fish liver from carcinogen damage and delays onset of malignancy. These findings indicate that transgenic fish models are powerful systems for investigating mechanisms of inhibition and regression of liver tumors.

In our earlier work, we demonstrated that agrin, a multifunctional heparan sulfate proteoglycan, accumulates in hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC). In addition, we proved the utility of agrin immumohistochemistry in discriminating between HCCs and benign parenchymal lesions. Here, we have examined the expression of agrin in metastatic liver carcinomas in comparison with primary liver tumors. Immunohistochemistry for agrin was performed on 25 HCC, 16 intrahepatic CCC, 20 colorectal cancer metastasis (CRCm), and 18 pancreatic ductal carcinoma metastasis (PDCm) samples and evaluated with both quantitative and qualitative methods. Agrin/CD34 double immunofluorescent staining was carried out on snap-frozen sections. Agrin mRNA expression was measured in 11 HCC, 7 CCC, 11 CRCm, and 12 normal liver tissues. Regardless of tumor grade, agrin immunostaining was strong in the microvessels of HCCs. As opposed to HCC, agrin immunostaining was faint or nearly absent from the CD34-positive microvessels of CCC, CRCm, and PDCm; rather, it was detected in the basement membranes surrounding tumor cell pseudoglandules. While agrin was preserved in the basement membranes of Grade III CCCs, it was nearly absent from poorly differentiated metastatic adenocarcinomas. Agrin mRNA levels were the highest in CCC and lower, but still elevated in HCC and CRCm. By qualitative evaluation of agrin immunoreactions, CCC was differentiated from CRCm and PDCm with a sensitivity of 0.81 and a specificity of 0.82. HCCs were unequivocally identified on the basis of microvascular agrin labeling. Thus, agrin immunohistochemistry may facilitate determination of primary versus metastatic origin in problematic liver cancer cases., (Copyright 2010 Elsevier Inc. All rights reserved.)

Unlabelled: The aim of the present study was to characterise the expression pattern of claudin-7 tight junction protein in canine normal liver, hyperplastic and primary neoplastic lesions of the canine liver and whether this tight junction protein can help differentiate canine cholangiocarcinomas from canine hepatocellular carcinomas., Methods and Results: Necropsy samples included 15 canine normal liver tissue samples, 10 hepatocellular nodular hyperplasias, 6 hepatocellular adenomas, 15 well-differentiated and 6 poorly differentiated hepatocellular carcinomas, 6 cholangiocellular hyperplasias, 10 cholangiocellular adenomas, 15 well-differentiated and 6 poorly differentiated cholangiocarcinomas, 6 normal extrahepatic bile ducts, 8 normal gall bladder tissue samples, and 5 cystic mucinous hyperplasias of the gall bladder. In all canine normal liver tissue samples the hepatocytes were negative for claudin-7 and the normal biliary epithelial cells showed intense basolateral membrane claudin-7 positivity. In all cholangiocellular hyperplasia samples and in all cholangiocellular adenoma samples the benign cholangiocytes showed intense basolateral membrane positivity for claudin-7. In all samples of the well-differentiated and poorly differentiated cholangiocarcinomas, the malignant neoplastic biliary epithelial cells showed intense basolateral membrane positivity for claudin-7. Neither the hyperplastic nodules of the liver cells nor the hepatocellular adenomas reacted with claudin-7. The well-differentiated and poorly differentiated hepatocellular cancers were negative for claudin-7. The epithelial cells of canine normal extrahepatic bile ducts, gall bladder and cystic mucinous hyperplasias of the gall bladder showed intense basolateral membrane positivity for claudin-7. Differences in the intensity of claudin-7 reaction were not apparent among different types of proliferative lesions of cholangiocytes or degrees of cellular differentiation of neoplastic biliary epithelial cells., Conclusion: Consequently, we hypothesize that claudin-7 is an excellent immunohistochemical marker of the cholangiocellular differentiation in canines and can be used to detect benign and malignant proliferative lesions of the canine biliary tract. It can also help to differentiate canine cholangiocarcinomas from hepatocellular carcinomas.

At selected sites around the UK, the offshore sentinel flatfish species dab Limanda limanda are found to contain elevated levels of macroscopic liver tumors. Previous proteomic and metabolomic studies have demonstrated that differences exist between tumor and non-tumor tissues; however, these differing features were not identified, and little is known about the changes at the gene expression level, or whether prognostic markers are present and can be identified. A flounder Platichthys flesus custom cDNA microarray and RT-PCR were used to investigate hepatic mRNA expression in the histologically confirmed tumors, hepatocellular adenoma (HA) and hepatocellular carcinoma (HC) from dab, and in adjacent normal tissue from the same fish. Differences in gene expression were observed between tumor and normal tissues, and between tumor types. A class-prediction approach using 50 transcripts revealed sufficient group-specific expression profiles to allow segregation of samples dependent on their tumor type or the sex of the host. Vitellogenins were found to display the greatest induction (up to 500-fold induction) in some HC tumors from female fish and in both HA and HC tumors from males. To the best of our knowledge, this is the first report of the association of vitellogenin expression with tumors of wild fish.

To search for a reliable biomarker of preneoplastic lesions arising early in mouse hepatocarcinogenesis the proteomes of microdissected basophilic foci, hepatocellular adenomas (HCAs), carcinomas (HCCs) and normal-appearing liver of B6C3F1 mice initiated with diethylnitrosamine (DEN) were analysed on anionic (Q10) surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) ProteinChip arrays. Significant overexpression of cytokeratin 8 (CK8; m/z 54, 565), cytokeratin 18 (CK18; m/z 47,538) proteins was found in basophilic foci as well as in HCAs and HCCs. Furthermore, immunohistochemistry demonstrated profound overexpression of CK8 and CK18 proteins (CK8/18) in all basophilic foci, mixed cell type foci, HCAs and HCCs in B6C3F1 and C57BL/6J mice initiated with DEN. A strong correlation between CK8/18-positive foci development and multiplicity of liver tumors in B6C3F1 and C57Bl/6J mice was further observed. Moreover, formation of CK8 and CK18 complexes due to CK8 phosphorylation at Ser73 and Ser431 was found to be strongly associated with neoplastic transformation of mice liver basophilic foci. Elevation of CK8/18 was strongly correlated with induction of cell proliferation in basophilic foci and tumors. In conclusion, our data imply that CK8/18 is a novel reliable marker of preneoplastic lesions arising during mouse hepatocarcinogenesis which might be used for prediction of tumor development and evaluation of environmental agents as well as drugs and food additives using mouse liver tests.

Background: Inflammatory hepatocellular adenoma (IHCA) defines a subgroup of hepatocellular adenomas characterized by the expression of members of the acute-phase inflammatory response [(serum amyloid A protein (SAA) and C-reactive protein (CRP)]. IHCA are unique or multiple as defined by the presence of several nodule(s) larger than 10 mm using both imaging and macroscopic observation. Frequently, additional micronodules (<10 mm), previously undetected by imaging, can be observed in resected specimens., Aims: To analyse micronodules in multiple (group 1, nine patients) and single (group 2, eight patients) IHCA cases, immunohistochemistry using SAA and CRP antibodies was performed on all nodules detected under macroscopic examination as well as on surrounding tissue with no visible nodules., Results: Nodules of different sizes (>5 < or = 10 mm, > or = 1 < or = 5 mm) were found in group 1, whereas only rare nodules in the mm range were found in group 2. Micronodules shared the characteristics of large nodules, which justified surgery such as inflammatory infiltrates, abnormal arteries, sinusoidal dilatation or peliosis. However, the number of these characteristics was proportional to the size of the nodules., Conclusion: This study demonstrates that the real number of IHCA is greater than that predicted from imaging-based analyses. In addition, we show that patients with more than one nodule present a greater chance to display more and larger undetected micronodules than patients with a single nodule.

Background/aims: Glycogen storage disease type Ib (GSD-Ib) patients deficient in a glucose-6-phosphate transporter (G6PT) manifest disturbed glucose homeostasis, myeloid dysfunctions, and hepatocellular adenoma (HCA). This study was conducted to evaluate whether maintaining normoglycemia in GSD-Ib could prevent HCA., Methods: We infused neonatal GSD-Ib mice with adeno-associated virus (AAV) carrying G6PT and examined their metabolic and myeloid phenotypes for the 72-week study., Results: The AAV vector delivered the G6PT transgene to the liver and bone marrow. Long-term metabolic correction was achieved alongside a transient myeloid correction. Hepatic G6PT activity was 50% of wild-type levels at 2 weeks post-infusion but declined rapidly thereafter to reach 3% of wild-type levels by age 6 to 72 weeks. Despite this, the infused mice maintained normoglycemia throughout the study, exhibited near normal growth and normalized serum metabolite profiles. However, all five AAV-treated GSD-Ib mice that lived over 50 weeks accumulated excessive hepatic glycogen and fat. Two mice developed steatohepatitis and multiple HCAs with one undergoing malignant transformation., Conclusions: Normoglycemia alone cannot prevent hepatic steatosis and glycogen accumulation or the development of HCAs in GSD-Ib, providing one explanation why GSD-Ib patients maintaining normoglycemia under intense dietary therapy continue at risk for this long-term complication.

We studied the tumor cell expression patterns of E-cadherin and matrix metalloproteinase-1, -2, -7, and -9 in a tissue microarray composed of 20 normal livers, 10 hepatocellular adenomas, 43 hepatocellular carcinomas with cirrhosis and 33 hepatocellular carcinomas without cirrhosis. Hepatocellular adenoma was characterized by the complete absence of matrix metalloproteinase-7 expression; hepatocellular carcinoma with cirrhosis was characterized by a significantly low expression of E-cadherin; and hepatocellular carcinoma without cirrhosis was characterized by low matrix metalloproteinase-9 expression. The staining intensity score of E-cadherin=3, matrix a metalloproteinase-7<1, and matrix metalloproteinase-9>or=2 can be used as the diagnostic criteria for hepatocellular adenoma and for distinguishing hepatocellular adenoma from normal, hepatocellular carcinoma with cirrhosis, and hepatocellular carcinoma without cirrhosis. E-cadherin<2 and matrix metalloproteinase-9<2 can be used for distinguishing both hepatocellular carcinoma with cirrhosis and hepatocellular carcinoma without cirrhosis from normal. Although statistically not significant, hepatocellular carcinoma without cirrhosis showed a higher E-cadherin expression and a lower matrix metalloproteinase-9 expression than hepatocellular carcinoma with cirrhosis, which could be partially responsible for the less aggressive behavior found in hepatocellular carcinoma without cirrhosis when compared with hepatocellular carcinoma with cirrhosis. These results, if confirmed in a further study of small biopsy specimens and of histologically ambiguous cases, could lead to the application of these markers in the diagnosis and differential diagnosis of hepatocellular neoplasms in our surgical pathology practice.

Unlabelled: We took advantage of the reported genotype/phenotype classification to analyze our surgical series of hepatocellular adenoma (HCA). The series without specific known etiologies included 128 cases (116 women). The number of nodules varies from single, <5, and >or=5 in 78, 38, and 12 cases, respectively. The resection was complete in 95 cases. We identified 46 HNF1alpha-inactivated HCAs (44 women), 63 inflammatory HCAs (IHCA, 53 women) of which nine were also beta-catenin-activated, and seven beta-catenin-activated HCAs (all women); six additional cases had no known phenotypic marker and six others could not be phenotypically analyzed. Twenty-three of 128 HCAs showed bleeding. No differences were observed in solitary or multiple tumors in terms of hemorrhagic manifestations between groups. In contrast, differences were observed between the two main groups. Steatosis (tumor), microadenomas (resected specimen), and additional benign nodules were more frequently observed in HNF1alpha-inactivated HCAs (P < 0.01) than in IHCAs. Body mass index > 25, peliosis (tumor), and steatosis in background liver were more frequent in IHCA (P < 0.01). After complete resection, new HCAs in the centimetric range were more frequently found during follow-up (>1 year) in HNF1alpha-inactivated HCA. After incomplete resection (HCA left in nonresected liver), the majority of HCA remained stable in the two main groups and even sometimes regressed. Six patients of 128 developed hepatocellular carcinoma (HCC) (all were beta-catenin-activated, whether inflammatory or not)., Conclusion: There were noticeable clinical differences between HNF1alpha-inactivated HCA and IHCA; there was no increased risk of bleeding or HCC related to the number of HCAs; beta-catenin-activated HCAs are at higher risk of HCC. As a consequence, we believe that management of HCA needs to be adapted to the phenotype of these tumors.

The present study was performed to characterize molecular expression levels of preneoplastic and neoplastic lesions induced by beta-naphthoflavone (BNF), an aryl hydrocarbon receptor (AhR) agonist in rat hepatocarcinogenesis. Male F344 rats were initiated with an intraperitoneal injection of 200 mg/kg N-diethylnitrosamine, and two weeks later, they were fed a diet containing 0% or 1% BNF for twenty-eight weeks. All animals were subjected to a two-thirds partial hepatectomy at week 3 and sacrificed at week 30. Histopathologically, BNF increased the incidence and multiplicity of altered foci (1.7-fold and 3.3-fold) and hepatocellular adenomas (HCAs) (4.0-fold and 4.7-fold). Immunohistochemically, BNF increased the number of proliferating cell nuclear antigen (PCNA)-positive cells in altered foci (2.3-fold) and HCAs (6.7-fold) compared with the surrounding tissue and decreased the staining of cell cycle regulators (P21, C/EBPalpha). In addition, loss of reactivity for AhR-regulated (CYP1A1, CYP1B1) molecules and increased reactivity of Nrf-2-regulated (AKR7, GPX2) molecules were also observed in proliferative lesions. Furthermore, increased staining of histone deacetylase (HDAC1) in the nucleus was prominent in HCAs. The differential expression patterns were confirmed at mRNA levels by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. These results suggest that enhanced cell proliferation and protection against oxidative stress play an important role in BNF-induced hepatocarcinogenesis in rats.

Agrin is a recently identified proteoglycan component of vascular and bile duct basement membranes in the liver. The selective deposition of agrin in hepatocellular carcinoma (HCC) microvessels versus sinusoidal walls prompted us to investigate the utility of agrin immunohistochemistry (IHC) in detecting malignant hepatocellular lesions. We focused on the differential diagnostic problems often presented by hepatocellular adenomas (HCAs) and dysplastic nodules. IHC for agrin was performed on 138 formalin-fixed, paraffin-embedded surgical specimens from 93 patients, including cirrhotic liver tissues (25), focal nodular hyperplasia (10), large regenerative nodules (8), low-grade (23) and high-grade (7) dysplastic nodules, small HCC (8), HCC (27), and HCA (30). Agrin immunostaining was compared with that of CD34 and, in selected cases, to glypican-3. The combination of agrin and CD34 sensitively (0.94) and specifically (0.93) identified lesions judged previously as malignant by histology. The majority of benign lesions were clearly agrin-negative, whereas the strength and extent of agrin IHC faithfully reflected dysplasia in "atypical" HCAs and in high-grade dysplastic nodules. Malignant lesions were uniformly positive. In conclusion, as agrin is highly selective for tumor blood vessels, IHC for agrin facilitates the discrimination of benign and malignant hepatocellular lesions. Moreover, whereas glypican-3 in some HCCs may appear in few scattered cells only, agrin is diffusely deposited in virtually all malignant lesions, which may prove advantageous in the evaluation of small specimens such as core biopsies.

Cytokeratins 7 and 19 and neuronal cell adhesion molecule (CD56) are differentially expressed in the hepatocytes and biliary epithelium. CD34 is an endothelial marker that is expressed in hepatic sinusoids in conditions associated with altered vascular flow and neoplasms. Distinct staining patterns using these markers have been shown in resected specimens of focal nodular hyperplasia, telangiectatic focal nodular hyperplasia, and hepatic adenoma. The purpose of this study was to examine the diagnostic use of these markers in needle biopsies. Needle biopsies from focal nodular hyperplasia (n = 21), telangiectatic focal nodular hyperplasia (n = 2), and hepatic adenoma (n = 14) were included in the study. These cases represent typical examples of each entity that have been diagnosed on the basis of clinical, imaging, and histologic features. Corresponding resection specimens available in 9 cases were also included in the study for comparison. Immunohistochemical analysis was performed on 4-mum-thick formalin-fixed and paraffin-embedded sections using antibodies against cytokeratin 7, cytokeratin 19, neuronal cell adhesion molecule, and CD34. The staining patterns and intensity for each marker were analyzed in a blinded fashion, and the patterns were recorded as focal nodular hyperplasia-like, hepatic adenoma-like, or indeterminate for each case. Presence of normal tissue was also recorded in each case. The hepatic adenoma-like pattern is characterized by strong cytokeratin 7 positivity in hepatocytes in patches with a gradual decrease in the staining intensity as the cells differentiate toward mature hepatocytes. Hepatic adenomas lack bile ducts and ductules as highlighted by cytokeratin 7, cytokeratin 19, and neuronal cell adhesion molecule stains. The focal nodular hyperplasia-like pattern is characterized by milder and focal cytokeratin 7 staining of hepatocytes. Cytokeratin 7, cytokeratin 19, and neuronal cell adhesion molecule show a strong staining of bile ductules in the fibrous septa. Normal liver shows cytokeratin 7, cytokeratin 19, and neuronal cell adhesion molecule staining of bile ducts, whereas the hepatocytes are generally negative. Of the 21 focal nodular hyperplasia cases, 20 cases (95.2%) showed a focal nodular hyperplasia-like pattern, whereas 13 (92.2%) of 14 hepatic adenoma cases showed a hepatic adenoma-like pattern. Both cases of telangiectatic focal nodular hyperplasia showed a hepatic adenoma-like pattern. CD34 stain showed areas of diffuse endothelial staining in 2 cases of hepatic adenoma, 3 cases of focal nodular hyperplasia, and both cases of telangiectatic focal nodular hyperplasia, whereas the remaining cases showed staining of endothelial cells only in the inflow areas of the sinusoids. A mixed (diffuse and inflow) pattern of CD34 staining was seen in 1 focal nodular hyperplasia, 1 hepatic adenoma, and 2 telangiectatic focal nodular hyperplasia cases. For statistical analysis, the telangiectatic focal nodular hyperplasia were considered as variants of hepatic adenoma. The findings were found to be highly statistically significant (P < .05) for cytokeratin 7, cytokeratin 19, and neuronal cell adhesion molecule stains. An inflow staining pattern favors a diagnosis of focal nodular hyperplasia; however, overall, CD34 stain was not helpful in differentiating focal nodular hyperplasia and hepatic adenoma. Corresponding resection specimens (hepatic adenoma = 5, focal nodular hyperplasia = 2) showed staining patterns that were identical to the biopsy, whereas resections of the telangiectatic focal nodular hyperplasia cases showed both focal nodular hyperplasia and hepatic adenoma-like areas. Considering that telangiectatic focal nodular hyperplasia is now thought to be a variant of hepatic adenoma, the staining patterns correctly identified all cases, except one case each of focal nodular hyperplasia and hepatic adenoma. In summary, a combination of cytokeratin 7, cytokeratin 19, and neuronal cell adhesion molecule immunostains performed on needle biopsies of liver shows distinctive patterns similar to that of resection specimen. The stains, especially cytokeratins 7 and 19, are very helpful in distinguishing normal from lesional tissue, as well as hepatic adenoma from focal nodular hyperplasia, and could be diagnostically helpful in challenging cases. Prospective studies to evaluate use of these stains in challenging cases are needed to validate these findings.

Unlabelled: Hepatocellular carcinomas (HCCs) and bile duct carcinomas (BDCs) have a poor prognosis. Therefore, surveillance strategies including sensitive and specific serum markers for early detection are needed. Recently, Golgi Phosphoprotein 2 (GOLPH2) has been proposed as a serum marker for HCC, but GOLPH2 expression data in liver tissues was not available. Using tissue microarrays and immunohistochemistry, we semiquantitatively analyzed GOLPH2 protein expression in patients with HCC (n = 170), benign liver tumors (n = 22), BDC (n = 114) and normal liver tissue (n = 105). A newly designed sandwich enzyme-linked immunoassay (ELISA) was used to analyze GOLPH2 levels in the sera of patients with HCC (n = 62), hepatitis C virus (HCV) (n = 29), BDC (n = 10), and healthy control persons (n = 12). By immunohistochemistry 121/170 (71%) of HCC showed strong GOLPH2 expression, which was significantly associated with a higher tumor grade (P = 0.01). A total of 97/114 (85%) BDCs showed a strong GOLPH2 expression which proved to be an independent prognostic factor for overall survival (P < 0.05). Serum levels of GOLPH2 measured by ELISA were significantly elevated in patients with HCC with underlying HCV infection (median 18 mg/L, P < 0.05) and patients with BDC (median = 14.5 mg/L, P < 0.01) in comparison to healthy controls (median 4 mg/L)., Conclusion: GOLPH2 protein is highly expressed in tissues of HCC and BDC. GOLPH2 protein levels are detectable and quantifiable in sera by ELISA. In patients with hepatitis C, serial ELISA measurements in the course of the disease appear to be a promising complementary serum marker in the surveillance of HCC. GOLPH2 should be further evaluated as a serum tumor marker in BDC on a larger scale.

Anterior gradient-2 expression is critical in normal embryonic development. Aberrant expression of anterior gradient-2 in adult tissues has been linked to breast, prostate, esophageal, and pancreatic carcinoma. To define the role of anterior gradient-2 in primary hepatocellular neoplasms, we used tissue microarrays and examined protein expression in typical hepatocellular carcinomas (n = 44), fibrolamellar carcinomas (n = 12), and hepatic adenomas (n = 9). In nonneoplastic liver tissues, anterior gradient-2 was expressed in the septal-sized bile ducts and weakly in zone 3 hepatocytes in 11 (18%) of 61 cases. In tumors, anterior gradient-2 was overexpressed by only 1 (2%) of 44 hepatocellular carcinomas. In contrast, 6 (75%) of 8 fibrolamellar and 3 (75%) of 4 metastatic fibrolamellar carcinomas were positive. All 9 hepatic adenomas were negative. Further analysis of mRNA in fibrolamellar carcinomas identified 2 novel splice variants, but expression levels were very low. Sequencing of the anterior gradient-2 gene in fibrolamellar carcinomas identified several polymorphisms (refSNP Ids: rs6842, rs8071, rs1051905) but no mutations. In conclusion, anterior gradient-2 is overexpressed in the majority of fibrolamellar carcinomas but is only rarely overexpressed in hepatocellular carcinomas.

Background: It has been suggested that the altered iron metabolism in liver tumors, characterized by the iron-deficient phenotype, is of importance for tumor growth., Aim: This study was performed to elucidate the mechanisms underlying iron deficiency in liver tumors by examining how the liver tumor development affects the expression of iron metabolism-related genes., Methods: Iron metabolism reference values were analyzed in the sera of diethylnitrosamine-induced hepatocellular adenoma-bearing mice. Expression of iron metabolism-related genes was analyzed in adenomas and surrounding non-tumor tissues, and a subgroup of adenoma-bearing mice loaded with iron 72h before sacrifice., Results: Iron content of the adenoma tissues was 2.0-2.5-fold lower compared to surrounding and age-matched control tissues. There was no significant difference in serum iron levels between the adenoma-bearing and control mice, while the adenoma-bearing mice exhibited a 2.4-fold lower level of serum transferrin saturation. Expression of iron metabolism-related genes was dysregulated in the adenomas. Iron loading affected protein expression similarly in the adenomas and surrounding tissues suggesting that iron-responsive regulation of the proteins was not impaired. However, the mRNA expression for ceruloplasmin and divalent metal transporter 1 (DMT1) IRE(+) in the adenomas was altered independently of iron status, and the dysregulation may contribute to diminished iron content., Conclusion: These findings suggest that diethylnitrosamine-induced liver adenoma-bearing mice have abnormal iron metabolism and that dysregulation of iron metabolism-related genes contributes to iron deficiency in the adenomas.

Context: Histopathologic distinction between hepatocellular carcinoma (HCC) and benign hepatocellular mass lesions, particularly hepatocellular adenoma, can sometimes be challenging. The currently available ancillary tools are suboptimal in terms of sensitivity and specificity., Objective: To further characterize the diagnostic value of glypican-3 (GPC3), a cell surface proteoglycan that has recently been shown to be overexpressed in HCC, in the distinction between HCC and benign hepatocellular mass lesions., Design: A total of 221 surgically resected liver specimens were subjected to immunohistochemical staining using a monoclonal antibody specific for GPC3. These included 111 HCCs, 48 hepatocellular adenomas, 30 focal nodular hyperplasias, and 32 large regenerative nodules in the background of cirrhosis., Results: Cytoplasmic, membranous, and canalicular staining for GPC3 was detected in 84 (75.7%) of the 111 HCCs, among which, 61 (72.6%) of the 84 cases exhibited diffuse immunoreactivity. In contrast, none of the 110 cases of hepatocellular adenoma, focal nodular hyperplasia, and large regenerative nodule showed detectable GPC3 staining. Focal GPC3 immunoreactivity was detected in cirrhotic nodules in 11 (16.4%) of 67 HCC cases with a cirrhotic background, but no background staining was observed in the remaining 44 HCCs without cirrhosis. GPC3 expression in HCCs did not correlate with the size, differentiation, or stage of the tumors; the presence or absence of cirrhotic background; or the underlying etiologies., Conclusions: GPC3 is a specific immunomarker for HCC that can be used to distinguish HCC from benign hepatocellular mass lesions, particularly hepatocellular adenoma. However, the diagnosis of HCC should not rely entirely on positive GPC3 immunostaining because focal immunoreactivity can be detected in a small subset of cirrhotic nodules. In addition, GPC3 expression in HCC can also be focal, and thus, the lack of GPC3 staining does not exclude the diagnosis of HCC.

Differentiating focal nodular hyperplasia from hepatic adenoma can be challenging. Cytokeratin 7, neuronal cell adhesion molecule, and cytokeratin 19 are differentially expressed in hepatocytes, biliary epithelium, and possibly hepatic progenitor/stem cells. CD34 is known to have altered expression patterns in the hepatic endothelium in conditions associated with abnormal perfusion and in hepatocellular carcinoma. The purpose of this study was to examine the expression pattern of these markers in focal nodular hyperplasia and hepatic adenoma and assess their diagnostic use. Ten resection specimens each of hepatic adenoma and focal nodular hyperplasia (including a case of telangiectatic focal nodular hyperplasia) were selected for the study. Immunohistochemical analysis was performed using antibodies against cytokeratin 7, cytokeratin 19, neuronal cell adhesion molecule, and CD34 on formalin-fixed, paraffin-embedded sections from each case. The staining patterns and intensity for each marker were analyzed. In hepatic adenoma, the cytokeratin 7 stain revealed strong positivity in hepatocytes in patches, with a gradual decrease in the staining intensity as the cells differentiated towards mature hepatocytes. Although bile ducts were typically absent in hepatic adenoma, occasional ductules could be identified with cytokeratin 7 stain. In focal nodular hyperplasia, cytokeratin 7 showed strong staining of the biliary epithelium within the fibrous septa and staining of the peripheral hepatocytes of most lobules that was focal and weaker than hepatic adenoma. Cytokeratin 19 and neuronal cell adhesion molecule showed patchy and moderate staining in the biliary epithelium of the ductules in focal nodular hyperplasia. While in the hepatic adenoma, cytokeratin 19 showed only rare positivity in occasional cells within ductules, and neuronal cell adhesion molecule marked occasional isolated cells in the lesion. CD34 showed staining of sinusoids in the inflow areas (periportal areas) in both focal nodular hyperplasia and hepatic adenoma. One case of telangiectatic focal nodular hyperplasia revealed both hepatic adenoma-like and focal nodular hyperplasia-like staining patterns. Distinct cytokeratin 7, cytokeratin 19, and neuronal cell adhesion molecule staining patterns are seen in hepatic adenoma and focal nodular hyperplasia possibly suggest activation of different subsets of hepatic progenitor/stem cell and can be diagnostically useful.

Unlabelled: Hepatocellular adenomas (HCAs) are a group of benign tumors forming three molecular pathological subgroups: (1) hepatocyte nuclear factor 1alpha (HNF-1alpha)-inactivated, (2) beta-catenin-activated, and (3) inflammatory. Some HCAs present both beta-catenin activation and inflammation. We analyzed magnetic resonance imaging (MRI) data for correlations between features on imaging and pathological classification of HCAs. We included 50 cases for which pathology specimens were classified into three groups based on immunohistochemical staining. Two characteristic MRI profiles were identified corresponding to HNF-1alpha-inactivated and inflammatory HCAs. Fifteen HCAs were HNF-1alpha-inactivated. The corresponding lesions showed (1) diffuse signal dropout on T1-weighted chemical shift sequence due to steatosis, (2) isosignal or slight hypersignal on T2-weighted (T2W) images, and (3) moderate enhancement in the arterial phase, with no persistent enhancement in the portal venous and delayed phases. For the diagnosis of HNF-1alpha-inactivated HCA, the positive predictive value of homogeneous signal dropout on chemical shift images was 100%, the negative predictive value was 94.7%, the sensitivity was 86.7%, and the specificity was 100%. Twenty-three HCAs were inflammatory and showed (1) an absence or only focal signal dropout on chemical shift sequence; (2) marked hypersignal on T2W sequences, with a stronger signal in the outer part of the lesions, correlating with sinusoidal dilatation areas; and (3) strong arterial enhancement, with persistent enhancement in the portal venous and delayed phases. Marked hypersignal on T2W sequences associated with delayed persistent enhancement had a positive predictive value of 88.5%, a negative predictive value of 84%, a sensitivity of 85.2%, and a specificity of 87.5% for the diagnosis of inflammatory HCA., Conclusion: HNF-1alpha-mutated HCAs and inflammatory HCAs were associated with specific MRI patterns related to diffuse fat repartition and sinusoidal dilatation, respectively.

Glypican-3 is a heparin sulfate proteoglycan normally expressed in fetal liver and placenta, but not in normal adult liver. Preliminary studies have shown that glypican-3 can be useful for the diagnosis of hepatocellular carcinoma. We performed immunohistochemistry for glypican-3 on 80 resection cases of hepatocellular lesions to examine the utility of glypican-3 immunohistochemistry in hepatocellular carcinoma at two ends of the differentiation spectrum. Staining was compared to Hep Par 1 in poorly differentiated cases. Glypican-3 was expressed in 46 (79%) hepatocellular carcinomas (56, 83 and 89% of well, moderately and poorly differentiated respectively) and seven (64%) fibrolamellar carcinomas. Of the 16 well differentiated cases, 10 closely resembled adenoma and were diagnosed due to focal abnormalities and/or loss of reticulin. Glypican-3 expression was seen in 50% in this group. Hepatocellular carcinomas arising in cirrhotic liver were more likely to be glypican-3 positive (91 vs 57%, P=0.004). All hepatic adenomas and macroregenerative nodules were negative, and three (43%) high grade dysplastic nodules were positive. Focal staining was seen in regenerative nodules in four (11%) cirrhosis cases. Glypican-3 was significantly more sensitive than Hep Par 1 for diagnosis of poorly differentiated hepatocellular carcinomas (89 vs 63%, P=0.02). The difference was more significant when only cases with diffuse positive staining were considered (83 vs 21%, P<0.001). In conclusion, glypican-3 has high sensitivity for the diagnosis of hepatocellular carcinoma, but is less sensitive in the extremely well differentiated hepatocellular carcinoma and fibrolamellar variant of hepatocellular carcinoma. Caution should be exercised in using glypican-3 in biopsy specimens as cirrhotic nodules can show strong expression. Glypican-3 can be especially useful in the identification of poorly differentiated hepatocellular carcinoma as it has higher sensitivity compared to Hep Par 1.

Hepatic adenomas are benign neoplasms of the liver that occur in several well-defined clinical settings, but principally that of excess hormone exposure. They have a small but poorly characterized risk of malignant degeneration. The clinical presentation and pathological findings were reviewed for all hepatic adenomas resected between January 1, 2003 and July 1, 2006. Immunohistochemistry for p53, beta-catenin and alpha-fetoprotein (AFP) were performed on those cases with malignant transformation and exon 3 of beta-catenin was amplified and sequenced. A total of 17 hepatic adenomas were resected and 3 showed malignant transformation. All three cases were in women with an age range of 23-33 years. The clinical presentations were vague abdominal pain. Histologically, the malignant transformation occurred within otherwise typical hepatic adenomas. Two of three cases showed patchy atypia throughout the hepatic adenoma. The hepatocellular carcinoma arose as distinct nodules directly within the adenomas, effectively ruling out synchronous lesions. The hepatocellular carcinomas were unifocal in two cases and multifocal in one case with the greatest dimensions of the hepatocellular carcinoma being 2.5, 2.2, and 1 cm. Immunostains for AFP and beta-catenin were negative in both the hepatic adenomas and areas of hepatocellular carcinoma. p53 immunostaining was positive within the areas of malignant transformation in one case. No mutations or deletions were seen in exon 3 of the beta-catenin gene for either the adenomas or the carcinoma. In conclusion, two of the cases that developed hepatocellular carcinomas showed cytological atypia in the background adenoma. The hepatocellular carcinomas arose as distinct nodules within the adenomas. No common molecular pathway of hepatocellular carcinogenesis was observed by examining AFP, beta-catenin, and p53 immunostains and no beta-catenin mutations or deletions were found.

Glycogen storage disease type I (GSD I) is a metabolic disorder resulting from defects in the glucose-6-phosphatase system. Approximately 75% of adolescent and adult patients develop hepatocellular adenomas, which can lead to considerable morbidity and mortality. The pathogenesis of adenomas is unclear and the risk of developing adenomas in treated patients is uncertain. The objective of this study was to determine whether metabolic imbalance was related to the occurrence of adenomas in patients with GSD I, and to determine what specific biochemical pathways were involved. We performed a 1:1 case-control retrospective study; cases were GSD I patients with adenomas and controls were GSD I patients without adenomas. Controls and cases were matched according to age at diagnosis, age at adenoma detection, and gender. We investigated biochemical abnormalities indicative of metabolic balance and exogenous factors potentially related to the onset of adenomas in the two groups. We detected no significant differences in dietetic treatment, compliance to treatment, or biochemical parameters related to metabolic balance between the two groups. In conclusion, we were unable to identify any significant differences in metabolic balance between GSD I patients who developed adenomas and those who did not.

In order to provide more efficient transduction of plasmid siRNA into target cells, develop more susceptible transduction into cancer cell types, and more easily explore application in animal experiments, we examined development of an adenoviral vector-mediated siRNA expression system and inhibition of survivin gene expression to induce the growth and apoptosis of hepatocarcinoma cells. A system of adenoviral vector-mediated siRNA expression was constructed for the survivin gene. Survivin gene expression in HepG2 cells infected with recombinant adenovirus was detected by Western blot and RT-PCR, and apoptotic cells were investigated by FAC. Western blot analysis showed that the infection of adenovirus-mediated siRNA against survivin efficiently inhibited the expression of survivin in hepatocarcinoma cells with an inhibitory rate of 66.32%. Semi-quantitative RT-PCR showed that survivin gene mRNA transcription was reduced by nearly 72.34% with a peak at 72 h. The number of apoptotic cells increased. In conclusion, results demonstrated that this adenovirus-mediated siRNA system could serve as a useful tool for both basic research on the analysis of gene function and cancer therapy applications.

Objectives: Superoxide dismutase (SOD) is a key antioxidant enzyme, responsible for scavenging of superoxide anion - a precursor of all reactive oxygen species (ROS). ROS are implicated in many pathologies, particularly in tumor disease. The aim of our work was to evaluate SOD isoforms' activity and protein level changes in liver tumors., Design and Methods: Materials were obtained from patients with liver tumors and with liver cirrhosis diagnosed by routine histopathological examination. Activity and protein level of SOD were determined by means of the Beauchamp and Fridovich assay and by Western blot analysis., Results: Decreased activity of CuZnSOD and MnSOD and distinct differences in SOD isoforms' protein expression in liver cirrhosis were found. Results also showed higher protein level and activity of SOD isoforms in liver malignant tumors than in benign ones., Conclusions: Malignant tumors have a better antioxidant system than benign ones. Moreover, weakening of antioxidant mechanisms and accumulation of oxidative damage in cirrhotic liver could initiate liver carcinogenesis.