Monique Fabre

Selective repopulation of normal mouse liver by Fas/CD95-resistant hepatocytes

Hepatocyte transplantation might represent a potential therapeutic alternative to liver transplantation in the future; however, transplanted cells have a limited capacity to repopulate the liver, as they do not proliferate under normal conditions. Recently, studies in urokinase (uPA) transgenic mice and in fumarylacetoacetate hydrolase (FAH)-deficient mice have shown that the liver can be repopulated by genetically engineered hepatocytes harboring a selective advantage over resident hepatocytes. We have reported that transgenic mice expressing human Bcl-2 in their hepatocytes are protected from Fas/CD95-mediated liver apoptosis. We now show that Bcl-2 transplanted hepatocytes selectively repopulate the liver of mice treated with nonlethal doses of the anti-Fas antibody Jo2. FK 506 immunosuppressed mice were transplanted by splenic injection with Bcl-2 hepatocytes. The livers of female recipients were repopulated by male Bcl-2 transgenic hepatocytes, as much as 16%, after 8 to 12 administrations of Jo2. This only occurred after anti-Fas treatment, confirming that resistance to Fas-induced apoptosis constituted the selective advantage of these transplanted hepatocytes. Thus, we have demonstrated a method for increasing genetic reconstitution of the liver through selective repopulation with modified transgenic hepatocytes, which will allow optimization of cell and gene therapy in the liver.

Towards an international pediatric liver tumor consensus classification: proceedings of the Los Angeles COG liver tumors symposium

Liver tumors are rare in children, and their diagnoses may be challenging particularly because of the lack of a current consensus classification system. Systematic central histopathological review of these tumors performed as part of the pediatric collaborative therapeutic protocols has allowed the identification of histologic subtypes with distinct clinical associations. As a result, histopathology has been incorporated within the Children’s Oncology Group (COG) protocols, and only in the United States, as a risk-stratification parameter and for patient management. Therefore, the COG Liver Tumor Committee sponsored an International Pathology Symposium in March 2011 to discuss the histopathology and classification of pediatric liver tumors, and hepatoblastoma in particular, and work towards an International Pediatric Liver Tumors Consensus Classification that would be required for international collaborative projects. Twenty-two pathologists and experts in pediatric liver tumors, including those serving as central reviewers for the COG, European Société Internationale d’Oncologie Pédiatrique, Gesellschaft für Pädiatrische Onkologie und Hämatologie, and Japanese Study Group for Pediatric Liver Tumors protocols, as well as pediatric oncologists and surgeons specialized in this field, reviewed more than 50 pediatric liver tumor cases and discussed classic and newly reported entities, as well as criteria for their classification. This symposium represented the first collaborative step to develop a classification that may lead to a common treatment-stratification system incorporating tumor histopathology. A standardized, clinically meaningful classification will also be necessary to allow the integration of new biological parameters and to move towards clinical algorithms based on patient characteristics and tumor genetics, which should improve future patient management and outcome.

Conditional cell ablation by tight control of caspase-3 dimerization in transgenic mice

Studying the effects of the loss of a specific cell type is a powerful approach in biology. Here we present a method based on the controlled activation of the apoptotic machinery. We expressed a modified caspase-3-containing chemical inducer of dimerization (CID)-binding sites in the livers of transgenic mice. In the absence of CID, no liver injury was detectable, underlining the absence of leakage in our system. In contrast, injection of the CID produced activation of the chimeric caspase-3, which led to a dose-dependent pure hepatocyte ablation with subsequent regeneration. This method is effective in both growing and nongrowing cells, and is therefore applicable to a wide range of cells and tissues. Moreover, because apoptosis has been described in numerous pathological circumstances, this system is useful for generating mouse models of human disorders as well as for studying the recovery or regeneration of tissues after cell loss.

Transcriptional profiling of pure fibrolamellar hepatocellular carcinoma reveals an endocrine signature

Fibrolamellar hepatocellular carcinoma (FLC) is a rare subtype of liver cancer occurring mostly in children and young adults. We have shown that FLC comprises two separate entities: pure (p‐FLC) and mixed‐FLC (m‐FLC), differing in clinical presentation and course. We show that p‐FLCs have a distinct gene expression signature different from that of m‐FLCs, which have a signature similar to that of classical hepatocellular carcinomas. We found p‐FLC profiles to be unique among 263 profiles related to diverse tumoral and nontumoral liver samples. We identified two distinct molecular subgroups of p‐FLCs with different outcomes. Pathway analysis of p‐FLCs revealed ERBB2 overexpression and an up‐regulation of glycolysis, possibly leading to compensatory mitochondrial hyperplasia and oncocytic differentiation. Four of the sixteen genes most significantly overexpressed in p‐FLCs were neuroendocrine genes: prohormone convertase 1 (PCSK1); neurotensin; delta/notch‐like EGF repeat containing; and calcitonin. PCSK1 overexpression was validated by immunohistochemistry, yielding specific, diffuse staining of the protein throughout the cytoplasm, possibly corresponding to a functional form of this convertase. Conclusion: p‐FLCs have a unique transcriptomic signature characterized by the strong expression of specific neuroendocrine genes, suggesting that these tumors may have a cellular origin different from that of HCC. Our data have implications for the use of genomic profiling for diagnosis and selection of targeted therapies in patients with p‐FLC. (Hepatology 2014;59:2228–2237)

Intrahepatic bile duct damage in children with Kawasaki disease

Three children with Kawasaki disease had liver biopsies because of evidence of hepatic disease. Cholangitis or bile duct injury and proliferation were found. Similar damage to the hepatic ductular system may explain the hydrops of the gallbladder sometimes seen in this disease.

Selection of in vivo retrovirally transduced hepatocytes leads to efficient and predictable mouse liver repopulation.

Stable liver gene transfer is generally limited by the low efficiency of commonly used vectors. One way to circumvent this difficulty is to confer a selective advantage on transduced hepatocytes, allowing them to progressively repopulate the liver. We have used a strategy for in vivo selection and controlled amplification of a small percentage of retrovirally engineered hepatocytes. Using a bicistronic retroviral vector encoding a reporter gene and Bcl2, which confers on liver cells a survival advantage against the Fas apoptotic pathway, we demonstrate that 1.5% of initially transduced hepatocytes repopulate up to 85% of the liver after 10 injections of a Fas agonist antibody. Moreover, we show that the kinetics of liver repopulation is highly predictable. This system provides a general means of expanding at will engineered hepatocytes in vivo and offering the possibility to obtain a genetically modified liver expressing a gene of interest in a desired proportion of hepatocytes.

Can pancreatic neuroendocrine tumour biopsy accurately determine pathological characteristics

BACKGROUND Assessment of the pathological characteristics of pancreatic neuroendocrine tumours is crucial for appropriate management. We compared preoperative pathological data with surgical specimens for accuracy. METHODS Surgical patients with pancreatic neuroendocrine tumours who underwent preoperative endoscopic ultrasound-guided fine needle aspiration of the primary tumour or biopsy of liver metastasis were retrospectively included. Tumour differentiation and the Ki67 proliferation index on biopsies were compared with pancreatic specimens. RESULTS Fifty-seven patients were included. A preoperative biopsy of the primary tumour or of a liver metastasis was obtained in 48 and 9 patients respectively. Tumour differentiation was high in 98%, and poor in 2% on biopsy and high in 100% of surgical specimens. Ki67 index values were 0 (0-19) and 2 (0-15) on biopsy and surgical specimens (p=0.01). Correlation between preoperative and surgical findings was stronger for liver (r=0.62, p=0.001) than for pancreas (r=0.23, p=0.11). Correlation for pancreas varied according to the tumour pattern: solid (r=0.24, p=0.16), mixed (r=0.91, p=0.0036) or cystic (r=0.04, p=0.89). Tumour grade was different between pancreatic biopsies and surgical specimens, for grade 1 (63% vs 37%) and grade 2 (28% vs 72%), p=0.0007. CONCLUSIONS Tumour grade assessment is accurate in biopsies of liver metastases of pancreatic neuroendocrine tumours, while pancreatic fine-needle aspiration biopsies are less accurate.

Quantitative study of centrolobular hepatic fibrosis in alcoholic disease before cirrhosis

In an attempt to determine the importance of perivenular fibrosis (PVF) in alcoholic liver disease, we studied 71 liver biopsies using histological grading and a morphometric method. The histological grading used 7 variables which allowed us to classify the patients into 7 groups: controls, patients without alcoholic hepatitis but with steatosis, steato-fibrosis, portal fibrosis and patients with mild, moderate, or severe alcoholic hepatitis. The quantitative analysis examined 3 parameters: (1) The inner diameter of the terminal hepatic veins (THV). (2) The thickness of the THV rims, related to perivenular fibrosis (PVF). (3) Centrolobular fibrosis (CLF) which represented the association of perivenular and perisinusoidal centrolobular fibrosis. No changes in the inner diameter of the terminal hepatic veins was observed for the different groups except in the case of severe alcoholic hepatitis. This fact indicated the absence of veno-occlusive lesions in early stages of mild and moderate alcoholic disease. In severe alcoholic hepatitis, THV were destroyed by centrolobular scars and most of them were indistinguishable and unmeasurable. Of the 26 cases with steatosis (with or without portal fibrosis) only two cases with steatofibrosis showed perivenular fibrosis. In contrast, a significant increase in PVF and in CLF appeared in patients with alcoholic hepatitis. CLF is easier to quantify and more significative than PVF. Thus, it seems to us that CLF is a better indicator of the intensity of sclerosis and of the risk of developing cirrhosis than PVF alone.

Peripancreatic Paraganglioma: A Potential Diagnostic Challenge in Cytopathology and Surgical Pathology

Paragangliomas are rare neuroendocrine neoplasms arising in extra-adrenal chromaffin cells of the autonomic nervous system. In rare instances, paragangliomas present around and involve the pancreas, thereby mimicking one of the more common primary pancreatic lesions. These neoplasms present considerable diagnostic difficulty not only for the clinician and radiologist but also for the pathologist. We have collected a series of 9 peripancreatic paragangliomas clinically simulating a primary pancreatic lesion. The paragangliomas were diagnosed in 4 men and 5 women with an age range of 37 to 78 years (mean, 50 y). Patients presented clinically either with diffuse epigastric and abdominal pain (7 of 9, 78%) or with an incidental mass (2 of 9, 22%) discovered on routine radiographic imaging. All patients were found to have mass lesions suspicious for a primary pancreatic neoplasm on radiographic examination. The lesions were predominantly located in the body of the pancreas (5 of 9, 56%) and ranged in size from 5.5 to 17.0 cm (mean, 10.0 cm). Five of 9 (56%) neoplasms also demonstrated cystic change. Fine-needle aspiration (FNA) was performed on 6 cases; however, the diagnostic accuracy was low, with 3 of 6 (50%) neoplasms misdiagnosed as pancreatic neuroendocrine tumor (PanNET) (n=1), spindle cell neoplasm (n=1), or pseudocyst (n=1). In addition, 2 of 8 (25%) surgically resected tumors were misdiagnosed by the referring pathologist as a PanNET. Immunohistochemistry was performed on all cases, confirming the characteristic 2-cell populations: chief cells (synaptophysin positive and chromogranin A positive) and sustentacular cells (S-100 protein positive). Follow-up information was available for all patients and ranged from 2 months to 11.6 years (mean, 2.7 y). Three of 9 (33%) patients developed metastatic disease, and 2 of these 3 died of their disease at 2.8 and 4.6 years after diagnosis. In summary, in unsuspected cases, interpretation of FNA and surgical pathology resections can be diagnostically challenging. Awareness and proper recognition of this entity, including differential diagnosis, are imperative in establishing the correct diagnosis. Further, close follow-up of these cases should be considered because of the significant risk of metastatic disease.

A preclinical model of hepatocyte gene transfer: the in vivo, in situ perfused rat liver

Delivering retroviruses targeted to hepatocytes in vivo involves the injection of retroviruses directly into the portal vein. The aim of this work was to establish a clinically relevant system for retrovirus-mediated gene transfer in a new model of in vivo, in situ perfused rat liver and to study the transgene expression. At 24 h after partial hepatectomy, the liver was completely excluded from the splanchnic circulation using an extracorporeal shunt. Two independent normothermal, oxygenated perfusion systems were used. First, liver perfusion was carried out with a recirculating system (1 h). Culture supernatant containing retroviruses (1.5 × 108 ffu/ml, β-galactosidase gene) was used as perfusate. Then the liver perfusion was maintained for more 30 min in a single liver passage system using culture medium without retroviruses as perfusate. High hepatocyte transduction rates (up to 34.4%) were obtained. PCR analysis showed no provirus in extrahepatic organs. Viral titrations performed simultaneously (inflow and outflow liver lines) showed that after 1 h of perfusion (up to 30 successive liver passages) retroviruses were still detected in the liver outflow perfusate (up to 2.0 × 107 ffu/ml). Washing the liver for 30 min dramatically decreased the leakage of retroviruses in the outflow. In order to be of clinical use, the injection of retroviruses targeted to hepatocytes in vivo should be done while the liver is completely excluded from the splanchnic circulation to avoid any extrahepatic retrovirus diffusion.