Walter J. Hofmann

Drug-induced apoptosis in hepatoma cells is mediated by the CD95 (APO-1/Fas) receptor/ligand system and involves activation of wild-type p53.

Chemotherapeutic drugs are cytotoxic by induction of apoptosis in drug-sensitive cells. We investigated the mechanism of bleomycin-induced cytotoxicity in hepatoma cells. At concentrations present in the sera of patients during therapy, bleomycin induced transient accumulation of nuclear wild-type (wt) p53 and upregulated expression of cell surface CD95 (APO-1/Fas) receptor in hepatoma cells carrying wt p53 (HepG2). Bleomycin did not increase CD95 in hepatoma cells with mutated p53 (Huh7) or in hepatoma cells which were p53-/- (Hep3B). In addition, sensitivity towards CD95-mediated apoptosis was also increased in wt p53 positive HepG2 cells. Microinjection of wt p53 cDNA into HepG2 cells had the same effect. In contrast, bleomycin did not enhance susceptibility towards CD95-mediated apoptosis in Huh7 and in Hep3B cells. Furthermore, bleomycin treatment of HepG2 cells increased CD95 ligand (CD95L) mRNA expression. Most notably, bleomycin-induced apoptosis in HepG2 cells was almost completely inhibited by antibodies which interfere with CD95 receptor/ligand interaction. These data suggest that apoptosis induced by bleomycin is mediated, at least in part, by p53-dependent stimulation of the CD95 receptor/ligand system. The same applies to other anti-cancer drugs such as cisplatin and methotrexate. These data may have major consequences for drug treatment of cancer and the explanation of drug sensitivity and resistance.

Survival and recurrence after liver transplantation versus liver resection for hepatocellular carcinoma: a retrospective analysis.

OBJECTIVE This study compares the results of liver transplantation (LTx) and liver resection (LR) for hepatocellular carcinoma (HCC) to test the widespread hypothesis that LTx is the preferable approach for small HCCs. SUMMARY BACKGROUND DATA With respect to scarcity of donor organs and poor results, LTxs for large HCCs are obsolete. Small HCC transplantations have been reported to result in an excellent survival rate. However, the data of comparative studies are controversial. METHODS Patients who were treated curatively by LTx (n = 50) or LR (n = 52) for HCC were included in this retrospective study. Survival and freedom from recurrence were analyzed. Patients were stratified according to prognostic factors (pT classification, tumor size, number of tumor nodules, vascular infiltration, and cirrhosis). RESULTS Overall, after LTx and LR the 3-year survival rate and recurrence rate were not significantly different. In the Cox analysis, tumor size (p = 0.02) and vascular infiltration (p = 0.04) were independent variables after LTx, whereas after LR, none of the tested prognostic parameters was significant. With regards to recurrence, tumor size was the only independent factor, after both LTx and LR (p = 0.02, respectively). Directly comparing the two therapeutic approaches, a 3-year survival rate in pT 1/2, oligocentric (1-5 nodules), and oligocentric and small tumors proved to be superior after LTx. The recurrence rate after LTx was superior to LR in pT 1/2 and oligocentric tumors. Remarkably, for small (< or = 5 cm) tumors, LTx and LR resulted in a similar 3-year survival rate and freedom from recurrence. CONCLUSIONS According to our analysis, the oncological advantage of LTx compared with LR is questionable. This applies especially for small tumors. Superior results of LTx in early stage HCC and particularly in oligocentric tumors may be attributed to incorrect preoperative diagnosis. Nevertheless, LTx is a reasonable treatment for patients with early stage tumors if a LR is impossible because of tumor localization or poor functional hepatic capacity.

Effect of ursodeoxycholic acid on liver and bile duct disease in primary sclerosing cholangitis. A 3-year pilot study with a placebo-controlled study period.

Primary sclerosing cholangitis is a cholestatic disease of the liver characterized by progressive fibrotic inflammation and obliteration of the extra- and/or intrahepatic bile ducts. There is no effective therapy. We, therefore, studied the safety and efficacy of ursodeoxycholic acid in patients with primary sclerosing cholangitis with or without additional ulcerative colitis. In a 1-year ursodeoxycholic acid treatment period, which preceded the controlled study period, ursodeoxycholic acid was well tolerated in 22 of 24 patients with ulcerative colitis and in all three patients without ulcerative colitis. In two patients with ulcerative colitis the dose of 750 mg ursodeoxycholic acid/day led to diarrhea, but following reduction of the dose to 500 and 250 mg/day ursodeoxycholic acid was well tolerated. After 1 year of ursodeoxycholic acid treatment, 20 patients were randomly assigned to receive either ursodeoxycholic acid 750 mg/day or placebo. All of them finished a double-blind, placebo-controlled study period. During ursodeoxycholic acid treatment, the liver enzymes improved markedly. The difference in alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and gamma-glutamyltransferase between the placebo and ursodeoxycholic acid group was significant (p < 0.05). Following ursodeoxycholic acid treatment, pruritus and fatigue improved in half of the patients but the difference between the placebo and ursodeoxycholic acid group was not significant. According to the ethical guidelines, after 3 months of placebo treatment, the controlled study had to be discontinued because of a more than twofold increase of serum transaminases in 8/10 patients on placebo. After the end of the controlled study, all patients were continuously treated with ursodeoxycholic acid for up to 4 years.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the multidrug resistance proteins MRP2 and MRP3 in human hepatocellular carcinoma.

Treatment of hepatocellular carcinoma (HCC) by chemotherapy is often impeded by the intrinsic multidrug resistance (MDR) of this frequent primary cancer of the liver. The MDR phenotype can be caused by ATP‐dependent export of chemotherapeutic drugs across the plasma membrane being mediated by transporters of the MDR P‐glycoprotein family or of the multidrug resistance protein (MRP) family. To elucidate the role of MRP family members in HCC, we analyzed the expression and subcellular localization of MRP1 (ABCC1), MRP2 (ABCC2) and MRP3 (ABCC3); all 3 isoforms have been shown to confer resistance to chemotherapeutic drugs. Semiquantitative RT‐PCR demonstrated that MRP2 and MRP3 mRNA expression in HCC was at least 10‐fold higher than MRP1 mRNA expression. MRP2 immunostaining was observed in 87% (33/38) of HCC samples. MRP2 was localized in the plasma membrane in a polarized fashion, either in trabecular structures resembling the canalicular membrane or in the luminal membrane when cells had a pseudoglandular arrangement. MRP3 was detected in all samples examined (9/9) by RT‐PCR and by immunofluorescence microscopy. MRP3 was localized to the basolateral membrane of carcinoma cells. Double‐label immunofluorescence microscopy with antibodies specific for MRP2 or MRP3 indicated that carcinoma cells expressed both MRP isoforms simultaneously. When MRP1 was detected by immunofluorescence microscopy, it was localized on the intracellular membranes of carcinoma cells. Thus, plasma membrane expression of MRP2 and MRP3, but not of MRP1, can contribute to the MDR phenotype of HCC.

Histologic classification of thymic epithelial tumors: comparison of established classification schemes.

The object of our multicenter retrospective study was to compare the new histologic World Health Organization (WHO) classification and the classical histologic Bernatz classification in terms of interobserver agreement and prognostic importance. The influence of coexisting diseases was also analyzed using the Charlson score. We evaluated 218 patients from 5 different hospitals who were treated between 1967 and 1998. The statistical methods of analysis included Kaplan‐Meier estimates of survival curves and the application of Cox proportional hazards models to identify sets of prognostic factors for survival. Interobserver agreement was assessed by kappa coefficients. For both WHO and Bernatz classifications, interobserver agreement was good (weighted kappa > 0.87). However, the subdiversification of the “bioactive” WHO subgroup (B1, B2, B3) resulted in an interobserver agreement of only 0.49 within this group. In multivariable models, both the WHO classification and the Bernatz classification including carcinomas showed similar prognostic capabilities. The B3 type in the WHO classification and the predominantly epithelial type in the Bernatz classification had an intermediate prognostic ranking in comparison with the carcinomas and with the other subgroups. For both classifications, further simplification and subclassification into 3 subgroups led to classes with good discriminative power in respect to survival. In addition, very good interobserver agreement was observed in the simplified classifications. Comorbidity, sex, age of the patient and lymphofollicular hyperplasia had no major influence on overall survival. Both classifications showed similar prognostic power. Interobserver agreement of the type B subgroups was only moderate. By simplification of the classifications, subgroups with distinct survival could be identified.

Human hepatic preneoplasia : phenotypes and proliferation kinetics of foci and nodules of altered hepatocytes and their relationship to liver cell dysplasia

Abstract Foci of altered hepatocytes (FAH) represent preneoplastic lesions, as shown in various animal models of hepatocarcinogenesis, but their significance in the human liver has not been established. The cellular composition, size distribution and proliferation kinetics of FAH in 163 explanted and resected human livers with or without hepatocellular carcinoma (HCC) and their possible association with small-cell change of hepatocytes (SCC) were therefore studied. FAH, including glycogen-storing foci (GSF), mixed cell foci (MCF) and basophilic cell foci, were found in 84 of 111 cirrhotic livers, demonstrating higher incidences in cases with (29/32) than in those without HCC (55/79). FAH were observed more frequently in HCC-free cirrhosis associated with hepatitis B or C virus or chronic alcoholic abuse (high-risk group) (37/47) than in that due to other causes (low-risk group) (12/21). MCF, predominant in cirrhotic livers of the high-risk group, were more proliferative, larger and more often involved in formation of nodules of altered hepatocytes (39.3%) than were GSF (8.5%). The results suggest that the FAH are preneoplastic lesions, MCF being more advanced than GSF. Oncocytic and amphophilic cell foci were also observed, but their significance remains to be clarified. Two types of SCC, namely diffuse and intrafocal SCC, were identified, but only intrafocal SCC was found to be related to increased proliferative activity and more frequent nodular transformation of the FAH involved, suggesting a close association with progression from FAH to HCC.

Localization of the Wilson's disease protein in human liver.

BACKGROUND & AIMS Wilsons disease is an autosomal-recessive disorder of copper metabolism that results from the absence or dysfunction of a copper-transporting P-type adenosine triphosphatase that leads to impaired biliary copper excretion and disturbed holoceruloplasmin synthesis. To gain further insight into the role of the Wilsons disease protein in hepatic copper handling, its localization in human liver was investigated. METHODS By use of a specific antibody, localization of the Wilsons disease protein was studied in liver membrane fractions and liver sections by immunoblotting, immunohistochemistry, and double-label confocal scanning laser microscopy. RESULTS The 165-kilodalton protein, found by immunoblotting, was most abundant mainly in isolated plasma membrane fractions enriched in canalicular domains. Immunohistochemistry revealed intracellular punctuate staining of hepatocytes in certain regions of the liver, whereas a canalicular membrane staining pattern was observed in other regions. Double-labeling studies showed that in the latter regions the transporter is present mainly in vesicular structures just underneath the canalicular membrane that are positive for markers of the trans-Golgi network. A weak staining of the canalicular membrane, identified by staining for P-glycoprotein, was observed. CONCLUSIONS These results show that in human liver the Wilsons disease protein is predominantly present in trans-Golgi vesicles in the pericanalicular area, whereas relatively small amounts of the protein appear to localize to the canalicular membrane, consistent with a dual function of the protein in holoceruloplasmin synthesis and biliary copper excretion.

β-catenin accumulation and mutation of the CTNNB1 gene in hepatoblastoma

Hepatoblastoma is a rare malignant tumor of the liver that occurs in children at an average age of 2 to 3 years. Epidemiologic studies have shown an increased frequency of this tumor type in families affected by adenomatous polyposis coli. In addition to the epidemiologic data, molecular genetic studies suggest that inactivation of the APC tumor suppressor may be involved in hepatoblastoma tumorigenesis. A major function of APC is the downregulation of β‐catenin, a transcription‐activating protein with oncogenic potential. In an ongoing immunohistochemical study of β‐catenin expression in sporadic cases of tumor types that are associated with adenomatous polyposis coli, we observed increased β‐catenin levels in the cytoplasm and in the nuclei of three investigated hepatoblastomas. Sequencing of exon 3 of the β‐catenin gene (CTNNB1) revealed an activating mutation in one of the tumor samples. Our data indicate for the first time that β‐catenin accumulation may play a role in the development of hepatoblastoma and that activating mutations of the β‐catenin gene may substitute biallelic APC inactivation in this tumor type. Genes Chromosomes Cancer 25:399–402, 1999.

Thymic medullary cells expressing b lymphocyte antigens

A small subpopulation of human thymic medullary cells was found to express B cell-restricted and -associated antigens in various combinations. The cells were detected in fetal, juvenile, and adult thymi using indirect immunoenzymatic methods and monoclonal antibodies (MoAbs). Morphologically, they could be subdivided into small, round lymphoid cells, accounting for less than 1% of medullary lymphoid cells, and into a larger variant, even more infrequent in number and asteroid in shape because of short cytoplasmic processes. Immunophenotype (CD19+, CD20+, CD22+, CD37+, IgM+, IgD+) and morphology of the first cell type led to the conclusion that the lymphoid cells were B lymphocytes. The second, asteroid cell type constantly expressed CD20 and inconstantly expressed IgM, CD19, CD22, and CD37; they were often found to form rosettes with non-B lymphocytes. It can be concluded that a small number of B cells and asteroid cells of still uncertain origin, but expressing B cell-restricted antigens, are constitutive elements of the fetal and adult thymic medulla. It can be hypothesized that the asteroid cell might represent a novel type of thymic accessory cell and that the rosetting of non-B lymphocytes around this asteroid cell might simulate or in fact be the earliest B cell interaction of maturing T cells.

Carcinoid tumors of the thymus. An immunohistochemical study

Five carcinoid tumors of the thymus were screened immunohistochemically for the occurrence of neuropeptides (ACTH, calcitonin, calcitonin gene‐related peptide, cholecystokinin, gastrin, neurotensin, somatostatin, substance P), as well as of serotonin, chromogranin A, and neuron‐specific enolase. Most of the patients exhibited local sysmptoms evoked by growing tumor masses in the upper mediastinum without any clinical evidence of endocrine activity. Light and electron microscopic examination showed characteristic uniform large epithelial cells in polar or palisade arrangement, containing variable amounts of electron‐dense secretory granules. Only a few of the tested neuropeptide antisera reacted with the investigated tumors. Cholecystokinin‐immunoreactive cell populations were seen in all tumors. Expression of neurotensin could be observed in three neoplasms, two of which also exhibited ACTH immunoreactivity. Chromogranin A‐immunoreactive cells were found in two neoplasms. Neuron‐specific enolase showed strong staining in three tumors, one of the tumors also being immunoreactive for calcitonin. The results were confirmed by control reactions. Apart from the demonstration that conventional marker proteins are not reliable in identifying all carcinoid tumors, the present study proves that the visualization of neuropeptide‐immunoreactive cells in thymus carcinoids does not necessarily correspond to the manifestation of the clinical symptoms. Furthermore, each of the investigated neoplasms, as also known from other carcinoid tumors, appears to be able to produce more than one hormone.