Predictive Patterns of Glutamine Synthetase Immunohistochemical Staining in CTNNB1-mutated Hepatocellular Adenomas.

Some hepatocellular adenoma (HCA) subtypes are characterized by different CTNNB1 mutations, leading to different beta-catenin activation levels, hence variable immunostaining patterns of glutamine synthetase (GS) expression, and different risks of malignant transformation. In a retrospective multicentric study of 63 resected inflammatory (n=33) and noninflammatory (n=30) molecularly confirmed CTNNB1-mutated b-(I)HCA, we investigated the predictive potential of 3 known GS patterns as markers for CTNNB1 exon 3, 7/8 mutations. Pattern 1 (diffuse homogenous) allowed recognition of 17/21 exon 3 non-S45 mutated b-(I)HCA. Pattern 2 (diffuse heterogenous) identified all b-(I)HCA harboring exon 3 S45 mutation (20/20). Pattern 3 (focal patchy) distinguished 12/22 b-(I)HCA with exon 7/8 mutations. In exon 3 S45 and 7/8 mutations, both b-HCA and b-IHCA showed a GS+/CD34- rim with diffuse CD34 positivity in the center of the lesion. Interobserver reproducibility was excellent for exon 3 mutations. Comparative analysis of GS patterns with molecular data showed 83% and 80% sensitivity (b-HCA/b-IHCA) and 100% specificity for exon 3 non-S45. For exon 3 S45, sensitivity was 100% for b-(I)HCA, and specificity 93% and 92% (b-HCA/b-IHCA). For exon 7/8, sensitivity was 55% for both subtypes and specificity 100% and 96% (b-HCA/b-IHCA). Preliminary data from 16 preoperative needle biopsies from the same patients suggest that this panel may also be applicable to small samples. In surgically resected HCA, 2 distinct GS patterns can reliably predict CTNNB1 exon 3 mutations, which are relevant because of the higher risk for malignant transformation. The third pattern, although specific, was less sensitive for the identification of exon 7/8 mutation, but the GS+/CD34- rim is a valuable aid to indicate either an exon 3 S45 or exon 7/8 mutation.

Does Argininosuccinate Synthase 1 (ASS1) Immunohistochemistry Predict an Increased Risk of Hemorrhage for Hepatocellular Adenomas?

Hepatocellular adenomas (HCAs) often pursue an innocuous clinical course. Recent work has elucidated important subtypes of HCA and biomarkers to identify them, including HCA at an increased risk for malignant transformation. Another key complication of HCAs is the risk of spontaneous tumoral hemorrhage, which may be life-threatening. Identification of a predictive biomarker for this clinical complication would therefore be of clinical value. It has been suggested that Argininosuccinate Synthase 1 (ASS1) immunohistochemistry (IHC) identifies HCA with a high propensity for hemorrhage. The aim of our study was to validate ASS1 IHC as a predictive marker of hemorrhage. Eighty-nine HCAs were collected for ASS1 IHC and subtyped according to published criteria. Clinical records were examined for evidence of tumoral hemorrhage. Twenty-one (23.6%) HCAs were complicated by clinically detected hemorrhage and were more likely to be resected (P=0.0002). Hemorrhage complicated all WHO subtypes of HCA. There was no association between hemorrhage and HCA subtype (P=0.92). Neither the distribution of ASS1 expression nor the intensity of ASS1 expression compared to normal liver showed a significant association with hemorrhage (P=0.051 and 0.34). Interlaboratory comparison of 8 cases showed good agreement regarding the intensity (6/8 and 7/8) and distribution of staining (7/8 and 7/8) across 3 laboratories performing ASS1 IHC. In conclusion, all subtypes of HCA may be complicated by hemorrhage. ASS1 IHC expression did not correlate with hemorrhagic complications. Caution is prudent before routine implementation of ASS1 IHC in clinical practice.

Unclassified hepatocellular adenoma expressing ASS1 associated with inflammatory hepatocellular adenomas.

Three liver nodules were fortuitously discovered in a 30-year-old obese woman during a gynecological workup and resected. Two nodules (6 and 1.5 cm) with histological characteristics of inflammatory hepatocellular adenoma (HCA) were C reactive protein positive with normal expression of glutamine synthetase. The third 6 cm nodule had all the characteristics of an Unclassified HCA with an overexpression of Argininosuccinate Synthase 1 (ASS1) in the tumor compared to the non-tumoral liver. The non-tumoral liver was highly steatotic. Upon MRI review, two HCAs were identified as inflammatory HCAs based on specific criteria. The third HCA was different from the other two with the presence of peculiar intratumoral fluid cavities. This first report on the association between unclassified HCA expressing ASS1 and inflammatory HCA reinforces the concept that common factors are implicated in HCA subtypes genesis. ASS1 is an interesting immuno-marker to identify among unclassified HCA a subgroup with a high risk of bleeding. ASS1 overexpression decreases sharply the number of "true" unclassified HCA.

Hepatic CYP1A2 activity in liver tumors and the implications for preoperative volume-function analysis.

Dynamic liver function assessment by the [13C]methacetin maximal liver function capacity (LiMAx) test reflects the overall hepatic cytochrome P-450 (CYP) 1A2 activity. One proven strategy for preoperative risk assessment in liver surgery includes the combined assessment of the dynamic liver function by the LiMAx test, the volumetric analysis of the liver, and calculation of future liver remnant function. This so-called volume-function analysis assumes that the remaining CYP1A2 activity in any tumor lesion is zero. The here presented study aims to assess the remaining CYP1A2 activities in different hepatic tumor lesions and its consequences for the preoperative volume-function analysis in patients undergoing liver surgery. The CYP1A2 activity analysis of neoplastic lesions and adjacent nontumor liver tissue from resected tumor specimens revealed a significantly higher CYP1A2 activity (median, interquartile range) in nontumor tissues (35.5, 15.9-54.4 µU/mg) compared with hepatocellular adenomas (7.35, 1.2-32.5 µU/mg), hepatocellular carcinomas (0.18, 0.0-2.0 µU/mg), or colorectal liver metastasis (0.17, 0.0-2.1 µU/mg). In nontumor liver tissue, a gradual decline in CYP1A2 activity with exacerbating fibrosis was observed. The CYP1A2 activity differences were also reflected in CYP1A2 protein signals in the assessed hepatic tissues. Volume-function analysis showed a minimal deviation compared with the current standard calculation for hepatocellular carcinomas or colorectal liver metastasis (<1% difference), whereas a difference of 11.9% was observed for hepatocellular adenomas. These findings are important for a refined preoperative volume-function analysis and improved surgical risk assessment in hepatocellular adenoma cases with low LiMAx values. NEW & NOTEWORTHY The cytochrome P-450 (CYP) 1A2-dependent maximal liver function capacity test reflects the overall functional capacity of the liver. To which extent hepatocellular tumors harbor CYP1A2 activity and thus contribute to the maximal liver function capacity test outcome is unknown. We here show that hepatocellular adenomas but not hepatocellular carcinomas or colorectal liver metastasis contain significant residual CYP1A2 activity. These findings are important for an improved preoperative volume-function analysis and an accurate surgical risk assessment in hepatocellular adenoma cases.

Characterization of high- and low-risk hepatocellular adenomas by magnetic resonance imaging in an animal model of glycogen storage disease type 1A.

Hepatocellular adenomas (HCAs) are benign tumors, of which the most serious complications are hemorrhage and malignant transformation to hepatocellular carcinoma (HCC). Among the various subtypes of HCA, the ß-catenin-activated subtype (bHCA) is associated with greatest risk of malignant transformation. Magnetic resonance imaging (MRI) is an important tool to differentiate benign and malignant hepatic lesions, and preclinical experimental approaches may help to develop a method to identify MRI features associated with bHCA. HCAs are associated with various pathologies, including glycogen storage disease 1a (GSD1a). Here, we utilized a mouse model for GSD1a that develops HCA and HCC, and analyzed the mice in order to distinguish low-risk from high-risk tumors. Animals were scanned by MRI using a hepato-specific contrast agent. The mice were sacrificed after MRI and their lesions were classified using immunohistochemistry. We observed that 45% of the animals developed focal lesions, and MRI identified four different patterns after contrast administration: isointense, hyperintense and hypointense lesions, and lesions with peripheral contrast enhancement. After contrast administration, only bHCA and HCC were hypointense in T1-weighted imaging and mildly hyperintense in T2-weighted imaging. Thus, high-risk adenomas display MRI features clearly distinguishable from those exhibited by low-risk adenomas, indicating that MRI is a reliable method for early diagnosis and classification of HCA, necessary for correct patient management.

Argininosuccinate synthase 1 and periportal gene expression in sonic hedgehog hepatocellular adenomas.

Genetic alterations define different molecular subclasses of hepatocellular adenoma (HCA) linked with risk factors, histology and clinical behavior. Recently, Argininosuccinate Synthase 1 (ASS1), a major periportal protein, was proposed as a marker of HCA with a high risk of hemorrhage. We aimed to assess the significance of ASS1 expression through the scope of the HCA molecular classification. ASS1 expression was evaluated using RNAseq, quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Immunohistochemistry. ASS1 and glioma-associated oncogene 1 (GLI1) expression were analyzed in vitro after modulation of GLI1 expression. Using RNAseq in 27 HCA and five nontumor liver samples, ASS1 expression was highly correlated with GLI1 expression (P<0.0001, R=0.75). In the overall series of 408 HCA, ASS1 overexpression was significantly associated with sonic hedgehog HCA (shHCA) compared to other molecular subgroups (P<0.0001), suggesting that sonic hedgehog signaling controls ASS1 expression. GLI1 expression silencing by siRNA induced a downregulation of ASS1 in PLC/PFR5 and SNU878 cell lines. In 390 HCA, we showed that ASS1 expression belonged to the periportal expression program that was maintained in shHCA but down-regulated in all the other HCA subtypes. In contrast, HCA with ß-catenin activation showed an activation of a perivenous program. Despite the significant association between GLI1 and ASS1 expression, ASS1 mRNA expression was not associated with specific clinical features. At the protein level using immunohistochemistry, prostaglandin D synthase (PTGDS) was strongly and specifically overexpressed in shHCA. CONCLUSION: ASS1 is associated with sonic hedgehog activation as part of a periportal program expressed in shHCA, a molecular subgroup defined by INHBE-GLI1 gene fusion. (Hepatology 2018).

Glycogen storage disease type Ia mice with less than 2% of normal hepatic glucose-6-phosphatase-α activity restored are at risk of developing hepatic tumors.

Glycogen storage disease type Ia (GSD-Ia), characterized by impaired glucose homeostasis and chronic risk of hepatocellular adenoma (HCA) and carcinoma (HCC), is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC). We have previously shown that G6pc-/- mice receiving gene transfer mediated by rAAV-G6PC, a recombinant adeno-associated virus (rAAV) vector expressing G6Pase-α, and expressing 3-63% of normal hepatic G6Pase-α activity maintain glucose homeostasis and do not develop HCA/HCC. However, the threshold of hepatic G6Pase-α activity required to prevent tumor formation remained unknown. In this study, we constructed rAAV-co-G6PC, a rAAV vector expressing a codon-optimized (co) G6Pase-α and showed that rAAV-co-G6PC was more efficacious than rAAV-G6PC in directing hepatic G6Pase-α expression. Over an 88-week study, we showed that both rAAV-G6PC- and rAAV-co-G6PC-treated G6pc-/- mice expressing 3-33% of normal hepatic G6Pase-α activity (AAV mice) maintained glucose homeostasis, lacked HCA/HCC, and were protected against age-related obesity and insulin resistance. Of the eleven rAAV-G6PC/rAAV-co-G6PC-treated G6pc-/- mice harboring 0.9-2.4% of normal hepatic G6Pase-α activity (AAV-low mice), 3 expressing 0.9-1.3% of normal hepatic G6Pase-α activity developed HCA/HCC, while 8 did not (AAV-low-NT). Finally, we showed that the AAV-low-NT mice exhibited a phenotype indistinguishable from that of AAV mice expressing ≥3% of normal hepatic G6Pase-α activity. The results establish the threshold of hepatic G6Pase-α activity required to prevent HCA/HCC and show that GSD-Ia mice harboring <2% of normal hepatic G6Pase-α activity are at risk of tumor development.

Correlation of exon 3 ß-catenin mutations with glutamine synthetase staining patterns in hepatocellular adenoma and hepatocellular carcinoma.

The current clinical practice is based on the assumption of strong correlation between diffuse glutamine synthetase expression and ß-catenin activation in hepatocellular adenoma and hepatocellular carcinoma. This high correlation is based on limited data and may represent an oversimplification as glutamine synthetase staining patterns show wide variability in clinical practice. Standardized criteria for interpreting diverse glutamine synthetase patterns, and the association between each pattern and ß-catenin mutations is not clearly established. This study examines the correlation between glutamine synthetase staining patterns and ß-catenin mutations in 15 typical hepatocellular adenomas, 5 atypical hepatocellular neoplasms and 60 hepatocellular carcinomas. Glutamine synthetase staining was classified into one of the three patterns: (a) diffuse homogeneous: moderate-to-strong cytoplasmic staining in >90% of lesional cells, without a map-like pattern, (b) diffuse heterogeneous: moderate-to-strong staining in 50-90% of lesional cells, without a map-like pattern, and (c) patchy: moderate-to-strong staining in <50% of lesional cells (often perivascular), or weak staining irrespective of the extent, and all other staining patterns (including negative cases). Sanger sequencing of CTNNB1 exon 3 was performed in all cases. Of hepatocellular tumors with diffuse glutamine synthetase staining (homogeneous or heterogeneous), an exon 3 ß-catenin mutation was detected in 33% (2/6) of typical hepatocellular adenoma, 75% (3/4) of atypical hepatocellular neoplasm and 17% (8/47) of hepatocellular carcinomas. An exon 3 mutation was also observed in 15% (2/13) of hepatocellular carcinomas with patchy glutamine synthetase staining. The results show a modest correlation between diffuse glutamine synthetase immunostaining and exon 3 ß-catenin mutations in hepatocellular adenoma and hepatocellular carcinoma with discrepancy rates >50% in both hepatocellular adenoma and hepatocellular carcinoma. The interpretation of ß-catenin activation based on glutamine synthetase staining should be performed with caution, and the undetermined significance of various glutamine synthetase patterns should be highlighted in pathology reports.

Multiple ß-catenin mutations in hepatocellular lesions arising in Abernethy malformation.

An 18-year-old man underwent liver transplantation due to an Abernethy malformation associated with multiple hepatocellular nodules including one which was rapidly enlarging and was suspicious for malignant transformation. Analysis of the explanted liver showed a spectrum of multiple hepatocellular nodules ranging in appearance from focal nodular hyperplasia, hepatocellular adenoma and to a well-differentiated hepatocellular neoplasm borderline for hepatocellular carcinoma. Mutational analysis revealed wild-type ß-catenin expression in the background liver and some nodules, whilst different variants were present in other lesions irrespective of their morphological appearance. No telomerase reverse transcriptase (TERT) promoter mutation was identified. Abernethy malformations can lead to independent genetic events which can result in ß-catenin mutations associated with malignant transformation of hepatocellular nodules. When following up such patients, one must therefore have a high index of suspicion, particularly if radiological surveillance reveals a change in the nature of hepatic lesions.

Genomic profiling of hepatocellular adenomas reveals recurrent FRK-activating mutations and the mechanisms of malignant transformation.

Hepatocellular adenomas (HCA) are benign liver tumors predominantly developed in women using oral contraceptives. Here, exome sequencing identified recurrent somatic FRK mutations that induce constitutive kinase activity, STAT3 activation, and cell proliferation sensitive to Src inhibitors. We also found uncommon recurrent mutations activating JAK1, gp130, or ß-catenin. Chromosome copy number and methylation profiling revealed patterns that correlated with specific gene mutations and tumor phenotypes. Finally, integrative analysis of HCAs transformed to hepatocellular carcinoma revealed ß-catenin mutation as an early alteration and TERT promoter mutations as associated with the last step of the adenoma-carcinoma transition. In conclusion, we identified the genomic diversity in benign hepatocyte proliferation, several therapeutic targets, and the key genomic determinants of the adenoma-carcinoma transformation sequence.

Diagnostic utility and limitations of glutamine synthetase and serum amyloid-associated protein immunohistochemistry in the distinction of focal nodular hyperplasia and inflammatory hepatocellular adenoma.

Inflammatory hepatocellular adenoma can show overlapping histological features with focal nodular hyperplasia, including inflammation, fibrous stroma, and ductular reaction. Expression of serum amyloid-associated protein in inflammatory hepatocellular adenoma and map-like pattern of glutamine synthetase in focal nodular hyperplasia can be helpful in this distinction, but the pitfalls and limitations of these markers have not been established. Morphology and immunohistochemistry were analyzed in 54 inflammatory hepatocellular adenomas, 40 focal nodular hyperplasia, and 3 indeterminate lesions. Morphological analysis demonstrated that nodularity, fibrous stroma, dystrophic blood vessels, and ductular reaction were more common in focal nodular hyperplasia, while telangiectasia, hemorrhage, and steatosis were more common in inflammatory hepatocellular adenoma, but there was frequent overlap of morphological features. The majority of inflammatory hepatocellular adenomas demonstrated perivascular and/or patchy glutamine synthetase staining (73.6%), while the remaining cases had diffuse (7.5%), negative (3.8%), or patchy pattern of staining (15%) that showed subtle differences from the classic map-like staining pattern and was designated as pseudo map-like staining. Positive staining for serum amyloid-associated protein was seen in the majority of inflammatory hepatocellular adenomas (92.6%) and in the minority of focal nodular hyperplasia (17.5%). The glutamine synthetase staining pattern was map-like in 90% of focal nodular hyperplasia cases, with the remaining 10% of cases showing pseudo map-like staining. Three cases were labeled as indeterminate and showed focal nodular hyperplasia-like morphology but lacked map-like glutamine synthetase staining pattern; these cases demonstrated a patchy pseudo map-like glutamine synthetase pattern along with the expression of serum amyloid-associated protein. Our results highlight the diagnostic errors that can be caused by variant patterns of staining with glutamine synthetase and serum amyloid-associated protein in inflammatory hepatocellular adenoma and focal nodular hyperplasia.

Ptpn11/Shp2 acts as a tumor suppressor in hepatocellular carcinogenesis.

The human gene Ptpn11, which encodes the tyrosine phosphatase Shp2, may act as a proto-oncogene because dominantly activating mutations have been detected in several types of leukemia. Herein we report a tumor-suppressor function of Shp2. Hepatocyte-specific deletion of Shp2 promotes inflammatory signaling through the Stat3 pathway and hepatic inflammation/necrosis, resulting in regenerative hyperplasia and development of tumors in aged mice. Furthermore, Shp2 ablation dramatically enhanced diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) development, which was abolished by concurrent deletion of Shp2 and Stat3 in hepatocytes. Decreased Shp2 expression was detected in a subfraction of human HCC specimens. Thus, in contrast to the leukemogenic effect of dominant-active mutants, Ptpn11/Shp2 has a tumor-suppressor function in liver.

Targeted deletion of liver glucose-6 phosphatase mimics glycogen storage disease type 1a including development of multiple adenomas.

BACKGROUND AND AIMS: Glycogen storage disease type 1a (GSD1a) is an inherited disease caused by a deficiency in the catalytic subunit of the glucose-6 phosphatase enzyme (G6Pase). GSD1a is characterized by hypoglycaemia, hyperlipidemia, and lactic acidosis with associated hepatic (including hepatocellular adenomas), renal, and intestinal disorders. A total G6pc (catalytic subunit of G6Pase) knock-out mouse model has been generated that mimics the human pathology. However, these mice rarely live longer than 3 months and long-term liver pathogenesis cannot be evaluated. Herein, we report the long-term characterization of a liver-specific G6pc knock-out mouse model (L-G6pc(-/-)). METHODS: We generated L-G6pc(-/-) mice using an inducible CRE-lox strategy and followed up the development of hepatic tumours using magnetic resonance imaging. RESULTS: L-G6pc(-/-) mice are viable and exhibit normoglycemia in the fed state. They develop hyperlipidemia, lactic acidosis, and uricemia during the first month after gene deletion. However, these plasmatic parameters improved after 6 months. L-G6pc(-/-) mice develop hepatomegaly with glycogen accumulation and hepatic steatosis. Using an MRI approach, we could detect hepatic nodules with diameters of less than 1 mm, 9 months after induction of deficiency. Hepatic nodules (1 mm) were detected in 30-40% of L-G6pc(-/-) mice at 12 months. After 18 months, all L-G6pc(-/-) mice developed multiple hepatocellular adenomas of 1-10 mm diameter. CONCLUSIONS: This is the first report of a viable animal model of the hepatic pathology of GSD1a, including the late development of hepatocellular adenomas.

Expression of xenobiotic and steroid hormone metabolizing enzymes in hepatocellular tumors of the non-cirrhotic liver.

Hepatocellular adenomas (HCA) and some hepatocellular carcinomas (HCC) arise in the non-cirrhotic liver. Although the liver is involved in the metabolism of a huge number of exogenous and endogenous substances, little is known about the role of metabolic enzymes in the development of liver tumors in the absence of cirrhosis. We analyzed the expression of glutathione S-transferases (GST) and cytochrome P450 enzymes (CYP) in 23 HCA, 20 HCC, and 22 focal nodular hyperplasias (FNH) using immunohistochemistry. The liver tissue revealed consistent specific staining for GST alpha, CYP1A1, 1A2, 2E1, and 3A4. In HCA and HCC, GST alpha expression was significantly reduced (p<0.001 and 0.043). Reduced GST alpha expression was significantly associated with steatosis in HCA and HCC (n=12, p=0.006), but not in non-neoplastic liver tissue. CYP3A4 expression was also reduced in HCA and HCC (p=0.03 and 0.02), and this was correlated with diabetes mellitus type 2 (p=0.02). In conclusion, HCA and HCC revealed changes in the expression of certain metabolic enzymes as compared with the non-neoplastic liver tissue or FNH. Therefore, reduced expression of GST alpha and CYP3A4 may indicate specific metabolic defects in the tumor tissue characterizing subgroups of HCA and HCC.

Activity and protein level of CuZnSOD and MnSOD in benign and malignant liver tumors.

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.

Association of CYP1B1 germ line mutations with hepatocyte nuclear factor 1alpha-mutated hepatocellular adenoma.

Biallelic somatic mutations of TCF1 coding for hepatocyte nuclear factor 1alpha (HNF1alpha) are found in 50% of the hepatocellular adenoma (HCA) cases usually associated with oral contraception. In rare cases, HNF1alpha germ line mutations could also predispose to familial adenomatosis. In order to identify new genetic factors predisposing to HNF1alpha-mutated HCA, we searched for mutations in genes involved in the metabolism of estrogen. For 10 genes (CYP1A1, CYP1A2, CYP3A4, CYP3A5, COMT, UGT2B7, NQO1, GSTM1, GSTP1, and GSTT1), we did not find mutations nor differences in the allele distribution among 32 women presenting HNF1alpha-mutated adenomas compared with 58 controls. In contrast, we identified a CYP1B1 germ line heterozygous mutation in 4 of 32 women presenting HNF1alpha-mutated adenomas compared with none in 58 controls. We confirmed these results with the identification of four additional CYP1B1 mutations in a second series of 26 cases. No mutations were found in the control group, which was extended to 98 individuals, and only a known rare genetic variant was observed in two controls (P = 0.0003). We did an ethoxyresorufin O-deethylase assay to evaluate the functional consequence of the CYP1B1 mutations. We found reduced enzymatic activity in each CYP1B1 variant. In addition, an E229K CYP1B1 mutation was found in a woman with a germ line HNF1alpha mutation in a familial adenomatosis context. In this large family, all three patients with adenomatosis bore both HNF1 and CYP1B1 germ line mutations. In conclusion, our data suggested that CYP1B1 germ line-inactivating mutations might increase the incidence of HCA in women with HNF1alpha mutations.

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

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

Polychlorinated biphenyls as initiators in liver carcinogenesis: resistant hepatocyte model.

A modified Solt-Farber protocol was established to investigate the potential initiating activity of lower chlorinated polychlorinated biphenyl (PCB) congeners in rat liver. Two different studies were conducted in male Fisher 344 rats. PCBs investigated were PCB3, PCB12, PCB38, and PCB77 in study 1 and PCB15, PCB52, PCB77, and the combination of PCB52 and PCB77 in study 2. Rats were subjected to partial hepatectomy followed by a single dose of the suspected initiating agent, diethylnitrosamine, or vehicle. Two weeks later all groups received selection treatment consisting of three daily doses of 2-acetylaminofluorene (2-AAF) and then a single dose of carbon tetrachloride, followed by three additional daily treatments of 2-AAF via gavage. Rats were killed 2 weeks after the last treatment of 2-AAF, and the number and volume of gamma-glutamyltranspeptidase (GGT)-positive foci were determined. Among the PCBs tested, PCB3, PCB15, PCB52, and PCB77 significantly increased the number of GGT-positive foci per cm(3) of liver and per liver. Only PCB3 and PCB15 increased the volume fraction of GGT-positive foci. Histopathologic analysis of hematoxylin- and eosin-stained liver sections showed that rats with significantly increased GGT-positive foci also had extensive cellular alteration. This effect was not seen in nonselection groups. We conclude that, under the conditions and time courses of these experiments, several PCBs have initiating activity in male Fischer 344 rats.

Immunocytochemical investigation of catalase and peroxisomal lipid beta-oxidation enzymes in human hepatocellular tumors and liver cirrhosis.

A significant reduction of catalase activity, a peroxisomal marker enzyme, occurs in human hepatic neoplasias, but no information is available on other peroxisomal proteins. We have studied by means of immunohistochemistry four specific proteins of peroxisomes (catalase and three enzymes of lipid beta-oxidation) in human hepatocellular tumors of various differentiation grades from adenoma to anaplastic carcinoma. In all tumors, except the adenomas, the tumor cells contained fewer peroxisomes than extrafocal hepatocytes and the reduction of antigenic sites in the tumor types generally correlated with the degree of tumor dedifferentiation as assessed by classical histopathological criteria. Two poorly differentiated tumors had no detectable peroxisomes at all. There were no major differences in the intensities of the immunocytochemical staining for all four studied peroxisomal antigens in different tumors, suggesting that the neoplastic transformation affects the biogenesis of the entire organelle and not merely the individual peroxisomal enzyme proteins. Some tumors exhibited a distinct peripheral distribution of peroxisomes. In cases with associated liver cirrhosis, the hepatocytes in the adjacent liver showed marked peroxisome proliferation, forming large perinuclear aggregates, occupying occasionally the entire cytoplasm. Taken together, our observations indicate that peroxisomes are significantly altered in both hepatocellular tumors and liver cirrhosis and, thus, could be responsible for some of the metabolic derangements observed in those disease processes.

Dose-dependent induction of carcinomas and glutathione S-transferase placental form negative eosinophilic foci in the rat liver by di(2-ethylhexyl)phthalate after diethylnitrosamine initiation.

The dose-dependence of di(2-ethylhexyl)phthalate (DEHP) hepatocarcinogenicity was investigated in male F344 rats which were initially injected with diethylnitrosamine (200 mg/kg, i.p.) and subjected to partial hepatectomy at week 3. The animals were administered DEHP in the diet at concentrations of 30, 300, 3,000, or 12,000 ppm starting 2 weeks after the DEN injection for up to 46 weeks and killed at weeks 8, 24, 48 and 52. Additional groups were given clofibrate (3,000 ppm in diet) or basal diet instead of the DEHP diet. Incidences of hepatocellular carcinomas were 75% (9/12, P < 0.01) for 12,000 ppm, 10% (1/10) for 3,000 ppm, 7% (1/14) for 300 ppm, 0% (0/13) for 30 ppm, 15% (2/13) for clofibrate, and 8% (1/13) for the basal diet group at week 52, 4 weeks after cessation of chemical feeding. Development of glutathione S-transferase placental form (GST-P) positive foci was only slightly increased by clofibrate-administration at week 52 and consistently lower than the control level in the DEHP-treated groups after 24 weeks. In contrast, GST-P negative eosinophilic foci were dose-dependently increased in the more than 300 ppm DEHP and clofibrate treated groups. At the 30 ppm dose level, however, no morphological changes were apparent in the liver. Thus, the non-observed effect level regarding the promotional activity of hepatocarcinogenesis was demonstrated at 30 ppm, the effects being predictable on the basis of development of GST-P negative eosinophilic foci.