Hepatospecific MR contrast agent uptake on hepatobiliary phase can be used as a biomarker of marked ß-catenin activation in hepatocellular adenoma.

OBJECTIVES: To assess the value of hepatospecific MR contrast agent uptake on hepatobiliary phase (HBP) images to detect marked activation of the ß-catenin pathway in hepatocellular adenomas (HCAs). METHODS: This multicentric retrospective IRB-approved study included all patients with a pathologically proven HCA who underwent gadobenate dimeglumine-enhanced liver MRI with HBP. Tumor signal intensity on HBP was first assessed visually, and lesions were classified into three distinct groups-hypointense, isointense, or hyperintense-according to the relative signal intensity to liver. Uptake was then quantified using the lesion-to-liver contrast enhancement ratio (LLCER). Finally, the accuracy of HBP analysis in depicting marked ß-catenin activation in HCA was evaluated. RESULTS: A total of 124 HCAs were analyzed including 12 with marked ß-catenin activation (HCA B+). Visual analysis classified 94/124 (76%), 12/124 (10%), and 18/124 (14%) HCAs as being hypointense, isointense, and hyperintense on HBP, respectively. Of these, 1/94 (1%), 3/12 (25%), and 8/18 (44%) were HCA B+, respectively (p < 0.001). The LLCER of HCA B+ was higher than that of HCA without marked ß-catenin activation in the entire cohort (means 4.9 ± 11.8% vs. - 19.8 ± 11.4%, respectively, p < 0.001). A positive LLCER, i.e., LLCER ≥ 0%, had 75% (95% CI 43-95%) sensitivity and 97% (95% CI 92-99%) specificity, with a LR+ of 28 (95% CI 8.8-89.6) for the diagnosis of HCA B+. CONCLUSIONS: Hepatospecific contrast uptake on hepatobiliary phase is strongly associated with marked activation of the ß-catenin pathway in hepatocellular adenoma, and its use might improve hepatocellular adenoma subtyping on MRI. KEY POINTS: • Tumor uptake on hepatobiliary phase in both the visual and quantitative analyses had a specificity higher than 90% for the detection of marked ß-catenin activation in hepatocellular adenoma. • However, the sensitivity of visual analysis alone is inferior to that of LLCER quantification on HBP due to the high number of HCAs with signal hyperintensity on HBP, especially those developed on underlying liver steatosis.

Human cirrhosis: monoclonal regenerative nodules derived from hepatic progenitor cells abutting ductular reaction.

Cirrhosis is a premalignant condition leading to hepatocellular carcinoma. Cirrhotic nodules are surrounded by a rim of CK 7/CK19-positive biliary cells termed ductular reaction. Half of all regenerative cirrhotic nodules are thought to be monoclonal by studying the pattern of inactivation of the X-linked human androgen receptor gene (HUMARA). Using a new technique for lineage tracing in human liver based on the identification in the mitochondrial DNA of mutations in the cytochrome c oxidase (CCO) gene, the authors discovered that 20% of regenerative nodules were monoclonal; in addition they showed that hepatic progenitor cells within abutting CCO-deficient cells of the ductular reaction had the same mutations as the adjacent regenerative nodule, indicating a common cell origin. It is the first direct evidence that regenerative nodules in cirrhosis can be derived from hepatic progenitor cells.

Cirrhosis: what else?

When the term cirrhosis was coined two centuries ago by Laennec, it designates by definition an end stage irreversible liver disease. Nowadays this word encompasses a whole range of disorders including some degree of reversibility for the early stage. It is therefore of prime importance to define the stages of the fibrotic process, based on the integration of knowledge about liver structure and function. In addition to morphological data, modern imaging techniques coupled to non-invasive biomarkers will probably help to better define and denominate this heterogeneous entity.

[Expression of the elastic fibers components during the fœtal liver development]. / Expression des composants de la matrice élastique au cours du développement hépatique fœtal.

Elastic fibers are composed of microfibrils containing fibrillin-1 and an elastic component, elastin. Microfibrils may not be associated with elastin. In the adult liver, fibrillin-1 and elastin are coexpressed within the stroma and portal tracts vessel walls. Fibrillin-1 is expressed alone around the bile ducts and within the Disse space. There is little work that has studied the elastic fiber organization during the fœtal liver development. Here, we studied the expression of fibrillin-1 and elastin by immunohistochemistry on 20 cases of fœtal liver. During the development of the portal tract, the two components are coexpressed on interstitial elastic fibers and within vessel walls. Fibrillin-1 is expressed alone around the bile structures during their maturation. Unlike adult liver, fibrillin-1 is expressed on thin and very irregular microfibrils within the Disse space. Our study shows that the elastic matrix development in the portal tract follows the development of the different structures, notably biliary structures. In the Disse space, microfibrils are not continuous. Their maturation may be in relation with the change of the hepatic blood flow after birth.

Differential effects of inactivated Axin1 and activated beta-catenin mutations in human hepatocellular carcinomas.

Perturbations to the Wnt signaling pathway have been implicated in a large proportion of human hepatocellular carcinomas (HCCs). Activating beta-catenin mutations and loss of function mutations in Axin1 are thought to be functionally equivalent. We examined the Wnt pathway in HCC by comparing the expression of beta-catenin target genes and the level of beta-catenin-dependent transcriptional activation, in 45 HCC tumors and four cell lines. Among these samples, beta-catenin and AXIN1 were mutated in 20 and seven cases, respectively. We found a significant correlation between activated beta-catenin mutations and overexpression of mRNA for the target genes glutamine synthetase (GS), G-protein-coupled receptor (GPR)49 and glutamate transporter (GLT)-1 (P=0.0001), but not for the genes ornithine aminotransferase, LECT2, c-myc and cyclin D1. We also showed that GS is a good immunohistochemical marker of beta-catenin activation in HCC. However, we observed no induction of GS, GPR49 or GLT-1 in the five inactivated Axin1 tumors. Beta-catenin-dependent transcriptional activation in two Axin1-mutated HCC cell lines was much weaker than in beta-catenin-mutated cell lines. Our results strongly suggest that in HCC, contrary to expectation, the loss of function of Axin1 is not equivalent to the gain of function of beta-catenin. Our results also suggest that the tumor suppressor function of Axin1 in HCC may be related to another, non-Wnt pathway.

Genomic alteration is not associated with fatty and clear cell change in hepatocellular carcinomas and its precursor nodular lesions in cirrhotic liver.

BACKGROUND: The aim of this study was to investigate whether fatty and clear cell areas in large regenerative nodules (LRN), dysplastic nodules (DN), and hepatocellular carcinoma (HCC) show higher degree of genomic mutation compared to non-fatty/clear cell area in the same nodule or non-lesional tissue. METHODS: We examined 22 nodular lesions (9 HCC, 5 DN and 8 LRN) from seven cirrhotic livers removed at transplantation. Frozen sections were used for manual microdissection of areas with fatty/clear cell change. DNA from microdissected tissue was amplified using arbitrarily primed polymerase chain reaction (AP-PCR), and PCR products were run on polyacrilamide gel generating a "fingerprint" band pattern. Autoradiographs were analysed using Adobe Photoshop version 6.0. Fingerprints from lesional tissue were compared to reference tissue and the total number of bands in excess or defect was calculated and divided by the total number of bands identified, obtaining the genomic damage fraction (GDF). RESULTS: Increasing GDF average values were seen from cirrhotic liver (0.13+/-0.04), to LRN (0.16+/-0.1), DN (0.28+/-0.08) and HCC (0.30+/-0.07). A statistically significant difference in GDF values was documented between cirrhotic liver and DN (p=0.008) and HCC (p=0.005) and between HCC and LRN (p=0.02). No significant difference was documented between DN and HCC, and between LRN and cirrhotic liver. Eleven nodules containing fat/clear cell areas were compared to the other 11 nodules without fat/clear cell areas. The GDF was not different between the two groups: 0.29+/-0.11 versus 0.25+/-0.12; p=0.5. The average value of genomic damage fraction between fat/clear cell areas (0.29+/-0.11) and no fat/clear cell areas (0.25+/-0.1) within the same nodules were not significantly different (p=0.11). CONCLUSION: Fatty and clear cell change in nodular lesions in cirrhotic liver may be an epigenetic phenotypic modification caused by microenvironmental factors such as ischaemia rather than indicating areas of increased malignant potential per se.

Tumor progression is associated with a significant decrease in the expression of the endostatin precursor collagen XVIII in human hepatocellular carcinomas.

Endostatin inhibits angiogenesis and tumor growth in mice. The role of its endogenous precursor collagen XVIII in human cancer is unknown. In normal tissues, two variants of collagen XVIII, namely, the short and long forms regulate tissue specificity, the long form being almost exclusively expressed by hepatocytes in the liver. We analyzed RNA arrays from 57 hepatocellular carcinomas (HCCs) with common and variant-specific probes and investigated the relationships between collagen XVIII expression and angiogenesis by measuring the CD34-positive microvessel density. Low collagen XVIII expression by tumor hepatocytes was associated with large tumor size (r, -0.63; P < 0.001) and replacement of trabeculae with pseudoglandular-solid architecture (chi2, 28; P < 0.001), which indicate tumor progression. Tumors expressing the highest collagen XVIII levels were smaller and had lower microvessel density (P = 0.01) than those expressing moderate levels; and HCCs with the lowest collagen XVIII levels approached a plateau of microvessel density, which indicated that a decrease in collagen XVIII expression is associated with angiogenesis in primary liver cancer. HCCs recurring within 2 years of resection showed 2.2-fold lower collagen XVIII mRNA than nonrecurring ones (P = 0.02). The findings relied on the hepatocyte-specific long form. Thus, the endogenous expression of the endostatin precursor decreases along with tumor progression in HCCs.

Concerted nonsyntenic allelic losses in hyperploid hepatocellular carcinoma as determined by a high-resolution allelotype.

Although the occurrence of loss of genetic material in hepatocellular carcinoma (HCC) has been documented both by cytogenetic analysis and by monitoring of allelic losses, a global overview of the extent and frequency of deletion occurring throughout the genome is not yet available. To contribute to this information, DNAs extracted from flow-sorted aneuploid nuclei from HCC and matched normal DNAs were typed for 275 microsatellite loci that were distributed along the autosomes. An average of 190 (69%) informative loci per case were generated on 48 HCC. Complete loss of heterozygozity in the tumor DNA was observed for 15.6% of the typed loci. The chromosome segments that were most frequently affected by deletion were: 8p (60%), 17p (48%), 1p (44%), 4q (42%), 16p (40%), 16q (39%), 6q (35%), 9p (30%), and 13q (29%). On average, 8 of the 39 chromosome segments studied per tumor carried at least one locus that demonstrated loss of heterozygosity (ie., the fractional allelic loss was 0.21). Groups of concerted nonsyntenic losses were observed for 16p and 1p and for 16p and 4q. The location of putative tumor suppressor genes on the most frequently deleted regions was confirmed and, in some cases, refined.

Investigation of liver fibrosis in clinical practice.

The liver is composed of different hepatic fibrogenic cells: hepatic stellate cells, portal fibroblasts, fibroblasts of the Glisson capsule surrounding the liver and vascular smooth muscle cells and the second layer cells present around centrolobular veins. During liver disease, one or several populations of these cells are activated, transformed into myofibroblasts and secrete the extra-cellular matrix. There are markers to identify hepatic stellate cells either quiescent (CRBP-1) or activated (alpha-smooth muscle actin). Liver biopsy, the current "gold-standard" to estimate liver fibrosis cannot be used anymore as a "gold standard". Furthermore, it is a costly procedure with adverse effects feared by patients and clinicians. Alternative to liver biopsy using non-invasive-tests or technics include FibroTest-ActiTest, transient-elastography, hepatic vein transit time using contrast ultrasonography, magnetic resonance imaging. As a routine test, the FibroTest-ActiTest is a validated one for patients with chronic hepatitis C. The advantage of the non-invasive tests or technics is that they provide a rapid and quantitative estimation of fibrosis. With these new methods, it is possible to follow the progression of the disease and its regression either spontaneously or under treatment. In conclusion, clinicians have in their hands several painless tools to explore liver fibrosis that can be easily repeated.

Classification tool for the systematic histological assessment of hepatocellular carcinoma, macroregenerative nodules, and dysplastic nodules in cirrhotic liver.

AIM: To design a classification tool for the histological assessment of hepatocellular carcinoma (HCC), dysplastic nodules (DN), and macroregenerative nodules (MRN) in cirrhotic liver. METHODS: Two hundred and twelve hepatocellular nodules (106 HCC; 74 MRN; 32 DN) were assessed systematically, quantitatively, and semiquantitatively as appropriate for 10 histological features that have been described as helpful in distinguishing small HCC, DN, and MRN in cirrhotic livers. The data were analyzed by multiple correspondence analysis (MCA). RESULTS: MCA distributed HCC, DN, and MRN as defined by traditional histological evaluation as well as the individual histological variables, in a "malignancy scale". Based on the MCA data representation, we created a classification tool, which categorizes an individual nodular lesion as MRN, DN, or HCC based on the balance of all histological features (i.e., vascular invasion, capsular invasion, tumor necrosis, tumor heterogeneity, reticulin loss, capillarization of sinusoids, trabecular thickness, nuclear atypia, and mitotic activity). The classification tool classified most (83%) of a validation set of 47 nodules in the same way as the routine histological assessment. No discrepancies were present for DN and MRN between the routine histological assignment and the classification tool. Of 25 HCC assigned by routine assessment in the validation set, 8 were assigned to the DN category by the classification tool. CONCLUSION: We have designed a classification tool for the histological assessment of HCC and its putative precursors in cirrhotic liver. Application of this tool systematically records histological features of diagnostic importance in the evaluation of small HCC.

Cytokeratin 7 and 20 expression in cholangiocarcinomas varies along the biliary tract but still differs from that in colorectal carcinoma metastasis.

In the liver, the immunostaining of cytokeratins (CK) 7 and 20 has been used to distinguish usual peripheral cholangiocarcinomas (CC) and colorectal carcinoma metastasis (CRM). However, other subtypes of CC are not infrequent and may be particularly difficult to distinguish from CRM by histology and even immunohistochemistry. Therefore, 48 CC from different locations, either peripheral (n = 19), or nonperipheral, that is, from the large intrahepatic bile ducts, the hilum, and the extrahepatic bile ducts (n = 29), and with different cytoarchitectural patterns were tested for CK7 and CK20 and compared with 31 CRM. CC were positive for CK7 and CK20 in 96% and 70%, respectively, whatever the architecture and differentiation of the tumor. The labeling index (LI) of CK7 in CC was always high, whereas it was low or moderate for CK20. CK20-positive phenotype was significantly more frequent in nonperipheral than in peripheral CC (82% vs 47%; p = 0.007). CRM were all positive for CK20 with a high LI, and mostly negative (81%) for CK7. In conclusion, (1) the CK immunoprofile of CC varies according to the location of the tumor in the biliary tract, peripheral CC being more often CK7+/CK20-, and nonperipheral ones CK7+/CK20+; and (2) a decision tree based on CK20 LI and CK7 positivity allows the distinction of CRM and CC, even for the nonperipheral type.

Relevance of vertical growth pattern in thin level II cutaneous superficial spreading melanomas.

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Liver adenomatosis with granulomas in two patients on long-term oral contraceptives.

We report two cases of liver cell adenomatosis associated with granulomatous hepatitis, both developed in white women (52 and 39 years of age) on long-term oral contraceptives (for 18 and 12 years, respectively). The first patient underwent surgery for five hepatic tumors 1-7 cm in diameter; the other patient had a partial segmentectomy for a 4-cm hepatic nodule of the right lobe. In both cases, dissection of the liver showed many other diffuse and smaller nodules. Histologically, all tumors were benign liver cell adenomas, with cellular atypia in the largest tumor and associated in both cases with granulomatous hepatitis, with numerous noncaseating epithelioid and giant cell granulomas located either only in the tumors (case 1) or diffusely in the tumoral and nontumoral hepatic parenchyma (case 2). During follow-up, ultrasound showed new nodular lesions in case 2, whereas in case 1 evolution was uneventful. In estroprogestative-associated liver diseases, adenomas are common, but adenomatosis and granulomas are rare. An association of these latter two conditions would seem to be exceptional. The prognosis for adenomatosis remains uncertain.

Cystic smooth-muscle tumor of the liver and spleen associated with Epstein-Barr virus after renal transplantation.

Immunosuppression is known to favor the development of various types of tumors. After organ transplantation, the risk of lymphoproliferative disease, whether clonal or not, is particularly increased and clearly associated with Epstein-Barr virus infection. We report a case of an unusual large cystic tumor of the liver with satellite hepatic and splenic nodules occurring 4 years after renal transplantation. Radiologic examination showed a rich vascularization of the tumor. Light and electron microscopy of a surgical liver biopsy, completed by an immunohistochemical study, demonstrated a well-differentiated tumor of smooth-muscle origin. Using in situ hybridization, we showed large amounts of Epstein-Barr virus messenger RNAs within the tumor cells. In addition, Southern blot analysis revealed that viral DNA was present in the form of a single monoclonal episome within the tumor. The polymerase chain reaction analysis of the genomic DNA of tumoral cells also indicated a monoclonal pattern. At last, the tumor was shown to be of host origin. Six months later, and despite three courses of chemotherapy, the tumoral lesions were unchanged. This case underlines the role of Epstein-Barr virus infection in the development of unusual and clonal smooth-muscle tumors after organ transplantation. The evolution of these rare tumors is uncertain.

Hepatic angiomyolipoma: a clinicopathologic study of 30 cases and delineation of unusual morphologic variants.

Hepatic angiomyolipoma (AML) is frequently misdiagnosed. HMB-45 is a promising immunomarker for this tumor that leads to recognition of some AMLs with unusual morphology. The purpose of this collaborative study is to better define the morphologic variations of AML. Thirty AMLs were examined, including four biopsy specimens and two fine-needle aspirates. The diagnosis was confirmed by the presence of HMB-45-positive myoid cells. Almost half the cases were originally misdiagnosed as carcinomas or sarcomas. There was marked female predominance (25:5), and the mean age was 48.7 years (range 29-68). Three patients (10%) had evidence of tuberous sclerosis and all had renal AML. According to the line of differentiation and predominance of tissue components, the tumors was subcategorized into mixed, lipomatous (> or = 70% fat), myomatous (< or = 10% fat), and angiomatous type. The mixed type was the most common (11 resected cases), comprising sheets of epithelioid muscle cells admixed with islands of adipocytes, abnormal vessels, and frequently, hematopoietic cells. Six tumors (including three from biopsy specimens) were heavily fatty and showed predominantly adipocytes with epithelioid and short spindle myoid cells webbed between fat cells. Of 10 myomatous AMLs, five tumors showed a pure sinusoidal trabecular pattern and comprised mainly epithelioid cells. Typically, mature adipocytes were absent or scanty, but fat was seen as fine droplets within cytoplasm or as occasional large globules in sinusoids. Pelioid and inflammatory pseudotumor-like patterns were identified focally. Regarding cellular features of the myoid cells, most of the epithelioid cells were either eosinophilic or clear with spiderweb cell morphology. Three AMLs showed an almost purely oncocytic appearance with scanty fat. Large pleomorphic epithelioid cells existed as small foci. Spindle cells arranged in long fascicles were uncommon. D-PAS-positive globules were common around pelioid areas. Brown pigments with staining characteristics of hemosiderin and/or melanin were noted. In conclusion, we propose HMB-45-positive myoid cells as the defining criterion of hepatic AML, which is a tumor capable of dual myomatous and lipomatous differentiation and melanogenesis. Because of its protean morphologic appearance, recognition of the various variant patterns and cell types is important for a correct diagnosis, assisted by immunohistochemical confirmation with HMB-45. Trabecular and oncocytic cell tumors appear to stand out as distinctive subtypes.

Trans-resveratrol, a grapevine-derived polyphenol, blocks hepatocyte growth factor-induced invasion of hepatocellular carcinoma cells.

We have shown that liver myofibroblasts stimulate in vitro invasion of hepatocellular carcinoma cell lines through a hepatocyte growth factor/urokinase-dependent mechanism. Resveratrol, a grapevine-derived polyphenol, has been shown to inhibit cellular events associated with tumor initiation, promotion and progression. The aim of this study was to evaluate the effects of trans-resveratrol on invasion of the human hepatoma cell line HepG2. Cell invasion was assessed using a Boyden chamber assay. Activation of the HGF signal transduction pathways was evaluated by Western blot with phospho-specific antibodies. Urokinase expression was measured by RT-PCR and zymography. Trans-resveratrol decreased hepatocyte growth factor-induced cell scattering and invasion. It also decreased cell proliferation without evidence for cytotoxicity or apoptosis. Trans-resveratrol did not decrease the level of the hepatocyte growth factor receptor c-met and did not impede the hepatocyte growth factor-induced increase in c-met precursor synthesis. Moreover, trans-resveratrol did not decrease hepatocyte growth factor-induced c-met autophosphorylation, or Akt-1 or extracellular-regulated kinases-1 and -2 activation. Finally, it did not decrease urokinase expression and did not block the catalytic activity of urokinase. In conclusion, our results demonstrate that trans-resveratrol decreases hepatocyte growth factor-induced HepG2 cell invasion by an as yet unidentified post-receptor mechanism.

Adenomatous hyperplasia in cirrhotic livers: histological evaluation, cellular density, and proliferative activity of 35 macronodular lesions in the cirrhotic explants of 10 adult French patients.

We examined 41 consecutive cirrhotic liver explants from French patients for the presence of nodules of adenomatous hyperplasia (AH) and then analyzed these lesions, together with underlying cirrhosis (C) and associated hepatocellular carcinoma (HCC), for various histological parameters, cellular density, and proliferative activity. Thirty-five AHs were identified in 10 livers (prevalence, 24%); seven of 10 were HCV positive. Hepatocellular carcinoma was more frequent in patients with AH than in patients without. The AHs consisted of 17 ordinary (OAH) and 18 atypical (AAH) adenomatous hyperplasia lesions. There was a malignant focus in five of the 18 AAHs. Wide areas of large liver cell dysplasia were frequent in OAH but never found in AAH. Obvious steatosis was frequent in HCC but exceptional in AAH and absent in OAH. There was a significant increase in cellular density in AAH and HCC as compared with C and OAH. Proliferative cell nuclear antigen immunostaining similarly showed an increase in proliferation from OAH or C to AAH and HCC. These data suggest that, in Europe as in Japan, one pathway of hepatocarcinogenesis is a multistep process in which AAH should be considered as a premalignant lesion very close to grade I HCC, while OAH seems to correspond to a regenerative nodule with limited proliferative ability.

Perisinusoidal fibrosis and basement membrane-like material in the livers of diabetic patients.

A direct correlation exists between collagenization of Disse's space and the presence of diabetic microangiopathy in type I diabetes. To confirm and extend this finding, we studied four liver biopsy samples from two patients with type I diabetes (one with retinopathy) and two patients with type II diabetes (no retinopathy). All had normal or subnormal results on liver function tests and normal liver architecture. Levels of collagen types I, III, and IV, laminin, and fibronectin, as determined by immunocytochemical techniques, appeared increased in all patients. Liver biopsy samples were perfusion fixed for electron microscopy of sinusoids and sinusoidal cells. Numerous and thick collagen bundles could be seen in Disse's space, as could the increase of basement membrane-like material underlying the endothelial cells, perisinusoidal cells, and sinusoidal membrane of hepatocytes. Perisinusoidal cells were active and had abundant rough endoplasmic reticula and thick processes. This preliminary study indicates that collagenization of Disse's space is not specific to a certain type of diabetes. The increase of basement membrane-like material raises the question of whether liver sinusoids are truly different from other capillaries as far as diabetic microangiopathy is concerned.

Longitudinal melanonychia in children. A study of eight cases.

BACKGROUND AND DESIGN: Longitudinal melanonychia is rare in white children and represents a difficult clinical challenge. Because of the fear of malignant melanoma, a surgical excision is usually performed, sometimes with definitive aesthetic and functional consequences. Eight children with longitudinal melanonychia who were between the ages of 2 and 14 years underwent follow-up. Surgical excision was performed in five cases. For three children, a wait-and-see policy was adopted, because their longitudinal melanonychia had been unchanged for years at the time of their examination. RESULTS: Melanoma was never observed in our cases (follow-up, 5.5 years). Histologic examinations performed in five cases showed junctional nevi of the nail matrix, often with dysplasia as commonly seen in juvenile nevi. Two children had postoperative nail dystrophy. CONCLUSION: In white children, longitudinal melanonychia rarely disappears. Since an ungual melanocytic band can appear at the age when other nevi appear, surgical excision should not be undertaken on different grounds than for other congenital or acquired nevi in children.