Louis Libbrecht

Hepatic stellate cell/myofibroblast subpopulations in fibrotic human and rat livers

BACKGROUND/AIMS Hepatic stellate cells (HSC) are commonly considered the precursor population of septal myofibroblasts (MF) in cirrhosis. We studied the distribution and expression profile of mesenchymal (myo)fibroblast-like populations in fibrotic and cirrhotic liver, in an attempt to elucidate their possible interrelationships. METHODS Fibrotic/cirrhotic livers (from 22 human explants and from two rat models: carbon tetrachloride intoxication, bile duct-ligation) were studied by means of immunohistochemistry (single and double immunostaining) with antibodies raised against desmin, alpha-smooth muscle actin (alpha SMA), glial fibrillary acidic protein (GFAP), neural-cell adhesion molecule (N-CAM), synaptophysin, neurotrophins, neurotrophin receptors and alpha B-crystallin (ABCRYS). RESULTS Septal MF showed the same expression profile as portal MF, in human and rat, being alpha SMA/ABCRYS/brain-derived nerve growth factor/GFAP-expression, with additional N-CAM- and desmin-expression in rat portal/septal MF. Perisinusoidally located HSC stained with all tested markers, MF at the septal/parenchymal interface showed an expression profile, intermediate between the profiles of HSC and portal/septal MF. CONCLUSIONS In advanced fibrosis and in cirrhosis, regardless of cause or species, three distinct mesenchymal (myo)fibroblast-like liver cell subpopulations can be discerned: portal/septal MF, interface MF and perisinusoidally located HSC. The fact that septal MF share more characteristics with portal MF than with HSC might suggest descent.

A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells

The variability in the prognosis of individuals with hepatocellular carcinoma (HCC) suggests that HCC may comprise several distinct biological phenotypes. These phenotypes may result from activation of different oncogenic pathways during tumorigenesis and/or from a different cell of origin. Here we address whether the transcriptional characteristics of HCC can provide insight into the cellular origin of the tumor. We integrated gene expression data from rat fetal hepatoblasts and adult hepatocytes with HCC from human and mouse models. Individuals with HCC who shared a gene expression pattern with fetal hepatoblasts had a poor prognosis. The gene expression program that distinguished this subtype from other types of HCC included markers of hepatic oval cells, suggesting that HCC of this subtype may arise from hepatic progenitor cells. Analyses of gene networks showed that activation of AP-1 transcription factors in this newly identified HCC subtype might have key roles in tumor development.

Morphological and biochemical characterization of a human liver in a uPA‐SCID mouse chimera

A small animal model harboring a functional human liver cell xenograft would be a useful tool to study human liver cell biology, drug metabolism, and infections with hepatotropic viruses. Here we describe the repopulation, organization, and function of human hepatocytes in a mouse recipient and the infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) of the transplanted cells. Homozygous urokinase plasminogen activator (uPA)‐SCID mice underwent transplantation with primary human hepatocytes, and at different times animals were bled and sacrificed to analyze plasma and liver tissue, respectively. The plasma of mice that were successfully transplanted contained albumin and an additional 21 human proteins. Liver histology showed progressive and massive replacement of diseased mouse tissue by human hepatocytes. These cells were accumulating glycogen but appeared otherwise normal and showed no signs of damage or death. They formed functional bile canaliculi that connected to mouse canaliculi. Besides mature hepatocytes, human hepatic progenitor cells that were differentiating into mature hepatocytes could be identified within liver parenchyma. Infection of chimeric mice with HBV or HCV resulted in an active infection that did not alter the liver function and architecture. Electron microscopy showed the presence of viral and subviral structures in HBV infected hepatocytes. In conclusion, human hepatocytes repopulate the uPA+/+‐SCID mouse liver in a very organized fashion with preservation of normal cell function. The presence of human hepatic progenitor cells in these chimeric animals necessitates a critical review of the observations and conclusions made in experiments with isolated “mature” hepatocytes. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005;41:847–856.)

The clinicopathological and prognostic relevance of cytokeratin 7 and 19 expression in hepatocellular carcinoma. A possible progenitor cell origin

Aims:  Cytokeratin (CK) 7 and CK19 expression, present in hepatic progenitor cells (HPCs) and in cholangiocytes but not in normal hepatocytes, has been reported in some hepatocellular carcinomas (HCCs); however, the incidence and relevance of this expression in HCC in Caucasians is not known. Therefore, our aim was to study the occurrence and clinicopathological characteristics of HCC expressing CK7 and/or CK19 in 109 Caucasian patients.

Glypican-3 expression distinguishes small hepatocellular carcinomas from cirrhosis, dysplastic nodules, and focal nodular hyperplasia-like nodules.

Distinguishing small hepatocellular carcinoma (HCC) from other types of small focal lesions that occur in a cirrhotic liver can be difficult on the basis of morphologic features alone. We investigated whether the expression of glypican-3 (GPC3) could be an ancillary tool in the histopathologic diagnostic process. We performed immunohistochemistry for GPC3 on 16 low-grade dysplastic nodules, 33 high-grade dysplastic nodules, 13 focal nodular hyperplasia-like nodules, and 59 HCCs with a diameter less or equal to 3 cm present in the cirrhotic liver of 66 patients. Both resected lesions and lesions biopsied by needle were included and nonlesional cirrhotic parenchyma was also stained. In a subset of cases (23 samples of cirrhosis, 4 low-grade dysplastic nodules, 5 high-grade dysplastic nodules, 2 focal nodular hyperplasia-like nodules, and 18 HCCs), real time reverse transcriptase-polymerase chain reaction for GPC3 was performed. GPC3 expression was, both on immunohistochemistry and by real time reverse transcriptase-polymerase chain reaction, much higher in small HCCs than in cirrhosis and other types of small focal lesions, indicating that the transition from premalignant lesions to small HCC is associated with a sharp increase of GPC3 expression in a majority of cases. The sensitivity and specificity of a positive GPC3-staining for the diagnosis of HCC in small focal lesions was 0.77 and 0.96, respectively, in resected cases, and 0.83 and 1, respectively, for needle biopsies. Because the result of the staining was easily interpretable, immunohistochemistry for GPC3 is valuable ancillary tool in the histopathologic diagnosis of small focal lesions in cirrhosis.

High expression of MDR1, MRP1, and MRP3 in the hepatic progenitor cell compartment and hepatocytes in severe human liver disease

An increase in bile ductular structures is observed in diverse human liver diseases. These structures harbour the progenitor cell compartment of the liver. Since ATP‐binding cassette (ABC) transporters may have a cytoprotective role in liver disease, an immunohistochemical study was performed on human liver specimens from patients with primary biliary cirrhosis (PBC), chronic hepatitis C virus (HCV) infection, submassive cell necrosis, and normal liver. The expression of MDR1, MDR3, BSEP, MRP1, MRP2, and MRP3 was determined using specific antibodies. Dilution series were constructed to determine the critical staining level in order to estimate the factor of up‐regulation. In normal liver, hepatocytes showed canalicular staining for MDR3, BSEP, and MRP2. MDR1 stained the canalicular membrane of hepatocytes as well as that of cholangiocytes. MRP3 showed low immunoreactivity of bile duct epithelial cells and centrilobular hepatocytes only. Normal liver showed no immunoreactivity for MRP1. In diseased liver, the expression of MDR3, BSEP, and MRP2 was relatively stable. In PBC, HCV, and submassive necrosis, the expression levels of MDR1, MRP1, and MRP3 were increased. The strongest immunoreactivity was seen after submassive necrosis, where remaining islands of hepatocytes showed strong canalicular staining for MDR1 and MRP3. Regenerating bile ductules at the interface of portal tracts and necrotic areas stained intensely for MDR1, MRP1, and MRP3. In conclusion, MDR1, MRP1, and MRP3 are up‐regulated in hepatocytes in severe human liver disease. Strong MDR1, MRP1, and MRP3 reactivity is seen in regenerating human bile ductules. Copyright

Deep intralobular extension of human hepatic 'progenitor cells' correlates with parenchymal inflammation in chronic viral hepatitis: can 'progenitor cells' migrate?

Ductular reaction and putative progenitor cells (or ‘progenitor cells’), which are presumed to be the human counterpart of the oval cells in rat liver, have been discerned in various human liver diseases, including chronic viral hepatitis. Since in experimental models of chronic hepatitis the activation of oval cells is correlated with the inflammatory infiltrate, this study investigated whether there is a correlation in chronic viral hepatitis between the number of ‘progenitor cells’ extending into the lobule and the severity of parenchymal inflammation, on the one hand, and the extent of ductular reaction and the severity of interface hepatitis, on the other hand. Liver biopsies of 55 patients with chronic hepatitis B and/or C were used. The severity of parenchymal inflammation and of interface hepatitis was semiquantitatively graded on a haematoxylin and eosin‐stained paraffin section, while the number of ‘progenitor cells’ and the extent of the ductular reaction were assessed on a serial section stained for cytokeratin (CK) 7. In addition, more extensive phenotyping of ‘progenitor cells’ was performed on sections from frozen material from five patients, using antibodies against CK7, CK8, CK18, CK19, chromogranin‐A, and the rat oval cell marker OV‐6. The number of more centrally located ‘progenitor cells’ correlated significantly with the severity of the parenchymal inflammation, while the extent of the ductular reaction correlated significantly with the severity of interface hepatitis. These findings suggest that in chronic viral hepatitis, inflammation plays a role in ‘progenitor cell’ activation and its topography. In cases with moderate and severe lobular inflammation, ‘progenitor cells’ were strikingly scattered throughout the parenchyma and surrounded by intermediate hepatocyte‐like cells, suggesting their migration into the parenchyma and their differentiation towards the hepatocytic lineage. Copyright

Preneoplastic lesions in human hepatocarcinogenesis

Abstract: The early stages of hepatocarcinogenesis in human chronic liver diseases are characterized by the emergence of preneoplastic lesions of which some will eventually develop into hepatocellular carcinoma (HCC). Basic studies on the genetic and epigenetic alterations of these preneoplastic lesions may eventually lead to new therapeutic strategies. Clinicopathological studies are also important in order to determine optimal management of patients with a preneoplastic lesion. This article aims to provide a comprehensive review of the current concepts of preneoplastic lesion in chronic liver diseases. The microscopical small‐cell dysplastic focus is the smallest morphologically recognizable precursor lesion of HCC and therefore is a logical target of study to elucidate the earliest events in hepatocarcinogenesis. In contrast, large‐cell dysplasia is not a precursor lesion, but appears to be of clinical value because of its good predictive value for development of HCC. Dysplastic nodules (DNs) are macroscopically recognizable precursor lesions of HCC and high‐grade DNs (HGDNs) have a risk of malignant transformation. Detection of DNs and correct differentiation from small HCC (<2 cm) is sometimes difficult, especially when only imaging techniques are used. Additional clinicopathological studies on identification and optimal treatment of DNs are necessary. Molecular studies on HGDNs and small HCCs may yield much information on the genetic mechanisms involved in the transition from severe dysplasia to early malignancy. In contrast, currently available data indicate that (large) regenerative nodules do not represent a distinct step in hepatocarcinogenesis. Animal models will be helpful in the further unravelling of human HCC development, provided that studies are performed on models that are good representatives of human hepatocarcinogenesis. We propose three criteria by which good mimickers can be identified.

Central Role of c-Myc during Malignant Conversion in Human Hepatocarcinogenesis

Hepatocarcinogenesis is a multistage process in which precursor lesions progress into early hepatocellular carcinomas (eHCC) by sequential accumulation of multiple genetic and epigenetic alterations. To decode the molecular events during early stages of liver carcinogenesis, we performed gene expression profiling on cirrhotic (regenerative) and dysplastic nodules (DN), as well as eHCC. Although considerable heterogeneity was observed at the regenerative and dysplastic stages, overall, 460 differentially expressed genes were detected between DN and eHCC. Functional analysis of the significant gene set identified the MYC oncogene as a plausible driver gene for malignant conversion of the DNs. In addition, gene set enrichment analysis revealed global activation of the MYC up-regulated gene set in eHCC versus dysplasia. Presence of the MYC signature significantly correlated with increased expression of CSN5, as well as with higher overall transcription rate of genes located in the 8q chromosome region. Furthermore, a classifier constructed from MYC target genes could robustly discriminate eHCC from high-grade and low-grade DNs. In conclusion, our study identified unique expression patterns associated with the transition of high-grade DNs into eHCC and showed that activation of the MYC transcription signature is strongly associated with the malignant conversion of preneoplastic liver lesions.

Forkhead Box Protein P1 Expression in Mucosa-Associated Lymphoid Tissue Lymphomas Predicts Poor Prognosis and Transformation to Diffuse Large B-Cell Lymphoma

PURPOSE Gene expression profiling studies have reported upregulated mRNA expression of forkhead box protein P1 (FOXP1) in response to normal B-cell activation and high expression in a poor prognosis subtype of diffuse large B-cell lymphoma (DLBCL). Recently, it was also found that FOXP1 rearrangements and expression of its protein occur in mucosa-associated lymphoid tissue (MALT) lymphomas. In this study, we investigated FOXP1 expression in its relationship to morphology, genetic features, and prognosis in a series of 70 MALT lymphomas. PATIENTS AND METHODS All samples were morphologically reviewed and stained for FOXP1. Presence of structural and/or numeric aberrations of the FOXP1, BCL10, and MALT1 genes was investigated. For all patients, a complete clinical data set was collected. RESULTS We detected nuclear expression of FOXP1 in 20 of the 70 MALT lymphomas (nine of them featuring structural or numeric aberrations of the FOXP1 locus). FOXP1 positivity was confined to MALT lymphomas with poor clinical outcome (with impact of FOXP1 expression on relapse rate and disease-free survival). It was also found that MALT lymphomas with strong FOXP1 expression are at risk of transforming into an aggressive DLBCL of nongerminal center phenotype if they feature, in addition, a polymorphic histology and the presence of trisomy 3 and 18. CONCLUSION The data presented show that FOXP1 expression is an independent prognostic factor in MALT lymphomas. The data also support the hypothesis that a subgroup of nongerminal center DLBCLs (those marked by FOXP1 expression and trisomy 3 and 18) might represent a large-cell variant of MALT lymphomas.