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Lenvatinib is a multiple receptor tyrosine kinase inhibitor (TKI) approved for first-line treatment of patients with unresectable hepatocellular carcinoma (HCC). TKI are suspected of exacerbating muscle loss in patients with cancer. In this study, we analyze the role of muscle loss in patients with advanced HCC treated with lenvatinib. This is a retrospective analysis of a real-life cohort of 25 patients with advanced HCC who were treated with lenvatinib from 2018 to March 2021 in Germany. Patients were stratified for loss of skeletal muscle area during the first three months of lenvatinib therapy. Overall survival (OS), progression-free survival (PFS) and toxicity were analyzed for all patients, especially regarding loss of muscle before and during the first three months of therapy with lenvatinib. Three months after beginning of therapy with lenvatinib, a significant reduction of muscle mass was observed in 60% of patients (p = 0.035). Despite increase of loss of skeletal muscle, patients benefitted from lenvatinib in our cohort of patients in terms of OS and PFS and did not experience increased toxicity. Furthermore, muscle loss was not a negative predictor of survival in the univariate analysis (p = 0.675). Patients with advanced hepatocellular carcinoma experience muscle loss with lenvatinib therapy. However, despite progressive muscle loss, patients benefit from a therapy with lenvatinib in terms of OS and PFS without increased toxicity. However, assessment and prophylaxis of skeletal muscle status should be recommended during a therapy with lenvatinib., (© 2024. The Author(s).)

Aim: Gallium-68-labelled inhibitors of the fibroblast activation protein (FAPi) enable positron emission tomography/computed tomography (PET/CT) imaging of fibroblast activation. We evaluated if [ 68 Ga]Ga-DATA5m.SA.FAPi PET/CT is related to Ki-67 as a marker of tumour aggressiveness in patients with liver metastases of NET., Methods: Thirteen patients with liver metastases of a histologically confirmed NET who underwent PET/CT with [ 68 Ga]Ga-DATA5m.SA.FAPi, [18F]FDG and [ 68 Ga]Ga-DOTA-TOC were retrospectively analyzed. PET-positive liver tumour volumes were segmented for calculation of volume, SUVmax and PET-positive tumour fraction (TF). PET parameters were correlated with Ki-67., Results: FDG SUVmax correlated positively (rho = 0.543, p < 0.05) and DOTATOC SUVmax correlated negatively (rho = -0.618, p < 0.05) with Ki-67, the correlation coefficients were in the moderate range. There was no significant correlation between FAPi SUVmax and Ki-67 (rho = 0.382, p > 0.05). FAPi TF correlated positively (rho = 0.770, p < 0.01) and DOTATOC TF correlated negatively (rho = -0.828, p < 0.01) with Ki-67, both significantly with high correlation coefficients. FDG TF also correlated significantly with Ki-67, with a moderate correlation coefficient (rho = 0.524, p < 0.05). The ratio FAPi VOL :DOTATOC VOL showed a significant and strong correlation with Ki-67 (rho = 0.808, p < 0.01)., Conclusion: The ratio FAPi VOL :DOTATOC VOL might serve as a clinical parameter for the assessment of dedifferentiation and aggressiveness of liver metastases in patients with NET. [ 68 Ga]Ga-DATA5m.SA.FAPi might hold potential for identification of high-risk patients. Further studies are warranted to evaluate its prognostic significance in comparison to [ 18 F]FDG in patients with NET., Competing Interests: Financial interests: RAB is a consultant for Bayer Healthcare (Leverkusen, Germany) and Eisai GmbH (Frankfurt, Germany). RAB has received speaker honorarium from Mediso Medical Imaging Systems Ltd., (Budapest, Hungary). ME is a consultant to Bayer, Novartis, Eisai and Ipsen. Non-financial interests: RAB has a non-commercial research agreement with Mediso Medical Imaging Systems Ltd., (Budapest, Hungary). Funding: The authors did not receive support from any organization for the submitted work., (Thieme. All rights reserved.)

Background: Interferon alpha (IFNalpha), currently used for the treatment of chronic viral hepatitis, is also known to prevent the development of hepatocellular carcinoma (HCC), the mechanism of this action being still debatable., Aims: To study thoroughly in human hepatoma cell lines (HHL)--Hep3B, HepG2, HuH7, SKHep1, and Chang-Liver--submitted to rhIFNalpha, the signalling pathway of IFNalpha, the binding activity of the cytokine on specific gamma-activated sequence (GAS) and interferon-stimulated regulatory element (ISRE) nuclear sequences, and its effects on apoptosis and cell proliferation., Methods: The behaviour of signal transducer and activator of transcription (STAT)1, STAT2, p48(IRF9) and the binding of nuclear proteins were investigated by immunoblot and electro-mobility shift assay. Expression of some IFNalpha-dependent proteins--p21/(WAF1), inducible nitric oxide synthase, IRF1 and 2--were studied by immunoblot. Apoptosis and the cell cycle were studied by morphological and biochemical methods., Results: Transduction of INFalpha was unaltered, although there were some variations in the different HHL. Nuclear protein binding to GAS or ISRE showed that ISRE was mainly involved. Apoptosis did not occur. The cell cycle was slightly modified in HuH7. Three GAS- and/or ISRE-dependent proteins increased, suggesting that IFNalpha may have some biological effects on HHL., Conclusions: The IFNalpha signalling pathway is functional in several HHL, but the cytokine has no apoptotic effect and a moderate anti-proliferative effect. This suggests that the preventive role of IFNalpha on HCC cannot be explained by an apoptotic and/or an anti-proliferative effect, but possibly by its action on several specific nuclear sequences that protect liver cells from transformation., (Copyright Blackwell Munksgaard 2004)

Background/aims: Fas-induced apoptosis is one of the main forms of apoptosis occurring in hepatocytes. We have previously demonstrated that the human hepatoma cell line Hep3B is resistant to Fas-mediated apoptosis. In this study, we investigated whether the human Fas receptor itself, or the Fas transduction pathway was responsible for the resistant phenotype., Methods: Clones of Hep3B cells overexpressing the mouse Fas gene (Hep3B(mfas)) were generated by transfection, and apoptosis was studied by (i) chromatin condensation and nuclear fragmentation, (ii) flow cytometry, (iii) DNA fragmentation and (iv) poly (ADP-ribose) polymerase cleavage., Results: Use of the species-specific and agonistic anti-mFas monoclonal antibody (JO2), showed that the mFas receptor was correctly routed to the plasma membrane of Hep3B(mfas) cells. Using the four above-mentioned criteria, we demonstrated that JO2 triggered mFas-mediated apoptosis of Hep3B(mfas), but not of Hep3B(pCi) cells (transfected with an empty vector)., Conclusions: Our data show (i) that the Fas signaling pathway can be completed when a functional mFas receptor is expressed in Hep3B cells, and thus, (ii) that the death-inducing signaling complex components and the effector caspases are functional in Hep3B cells. Moreover, they suggest that the Fas subunits are not pre-assembled at the cell membrane before receptor-ligand interaction.

The in vitro occurrence of apoptosis in hepatic cells has not been well characterized because it depends on apoptosis inducing-agents and culture conditions. Furthermore, for a given hepatic cell and the same agent, discrepant results have been reported depending on the technique used to evaluate the proportion of apoptotic cells. In this study, we compared the effects of several apoptosis-inducing agents - transforming growth factor beta1 (TGF-beta1), retinoic acid (RA), okadaic acid (OA), and cycloheximide (CY) - on two types of hepatic cells, the human hepatoma cell line Hep3B and normal rat hepatocytes, maintained either plated for 24 to 48 h or in suspension for 20 h. Chromatin condensation and/or nucleus fragmentation were investigated morphologically by DAPI staining. DNA fragmentation was investigated biochemically by agarose gel electrophoresis and poly(ADP-ribose) polymerase (PARP) cleavage was studied by western blot. Apoptotic cells were quantified either by counting cells on UV microscopy after DAPI staining or by flow cytometry. Nuclear changes, the ladder pattern on DNA electrophoresis and PARP cleavage were observed in plated cells, hepatoma cells and normal rat hepatocytes, with all inducers but especially with OA. Semiquantification confirmed that OA was a strong inducer in plated cells under the present conditions, since about 14% and 30% of Hep3B cells (with DAPI staining and flow cytometry, respectively) were apoptotic after 48 h treatment, while, with the other inducers, apoptosis was weaker and discrepancies were also observed between the two counting methods (TGF-beta1; 4% and 12%; RA, 7% and 12%; CY, 4% and 16%, with DAPI staining and flow cytometry, respectively). OA induced a moderate apoptosis in cultured hepatocytes (13% with DAPI staining), while TGF-beta1, RA and CY were found to be weakly apoptotic (respectively 4% for the first two and 6% for the last ) after 48 h. In contrast, in suspension cells, apoptosis was observed neither in Hep3B cells nor in normal hepatocytes, whatever the apoptotic inducer and whatever the techniques used to detect apoptosis. In conclusion, our results show that induction of apoptosis in hepatic cells depends not only on the apoptosis-inducing agent but also on the culture conditions.

Liver carcinogenesis is associated with striking changes in the integrin repertoire of hepatocytes, including the overexpression of the laminin and collagen receptors alpha1beta1 and the de novo induction of the laminin receptor alpha6beta1. Our aim was to analyze the role of pro-inflammatory cytokines, interferons and fibrogenic cytokines TGF-beta and FGF2 in the regulation of the expression of beta1 integrins by neoplastic hepatocytes. The 2 human hepatocellular cell lines HepG2 and Hep3B were used as models. Integrin expression was assessed by qualitative methods (immunocytochemistry, Western blotting) and semi-quantitative techniques (FACS, cellular ELISA), before and after stimulation by TNFalpha, IL1-beta, TGF-beta, FGF2, interferon gamma and interferon alpha-2b. HepG2 and Hep3B constitutively expressed alpha1, alpha2, alpha6 and beta1 chains. A 24 to 48-hr stimulation with pro-inflammatory cytokines, TGF-beta and FGF2 induced a significant increase in the concentrations of all integrin chains. The maximum induction was registered for beta1 chain, which presented increases amounting up to 3, 4 and 7 times the control values in the presence of, respectively, TNF alpha/IL1-beta, TGF-beta and FGF2. Interferons had no direct effect on integrin expression and partially antagonized the effects of TNF alpha and TGF-beta. The increased concentrations of integrin chains were associated with an increased membrane expression of the corresponding dimers and with an increased adhesion of stimulated hepatocytes to laminin, which was antagonized by neutralizing anti-beta1 and anti-alpha6 antibodies. Finally, anti-alpha6 antibody inhibited the migration of HepG2 and Hep3B cells in reconstituted basement membrane. Our results suggest that the stimulation of alpha6beta1 integrin expression in hepatocarcinoma cells is essential for cell adhesion and migration., (Copyright 1999 Wiley-Liss, Inc.)

Background/aims: Little is known about genetic alterations in large or small liver cell dysplasia. The aim of this study was to determine whether these lesions present numerical chromosome aberrations., Methods: Eight patients with hepatocellular carcinoma on cirrhosis, five with large liver cell dysplasia and three with small liver cell dysplasia, were analysed by in situ hybridization with different centromeric nucleic acid probes specific respectively for chromosomes 1, 7, 17 and 18. In each case results were compared between dysplastic, tumoral and non-dysplastic cirrhotic cells. Four normal livers were also studied with the same method and served as cytogenetic controls., Results: All cases of large liver cell dysplasia dysplayed a polysomic population for each investigated chromosome. A high variability of numerical chromosome aberrations was observed with a copy number of chromosomes which ranged from two to more than six. By contrast, only one case of small liver cell dysplasia showed chromosomal anomalies. Numerical aberrations of at least one chromosome were observed in six of the eight hepatocellular carcinoma while the non-dysplastic cirrhosis and normal liver always showed a diploid pattern., Conclusions: These results demonstrate that cellular modifications in large liver cell dysplasia coexist with an early acquisition of genomic alterations, supporting the view that these phenotypic changes are preneoplastic.

We have analyzed the composition of the tumor stroma and the expression of cell-matrix and cell-cell adhesion molecules in 11 cases of fibrolamellar carcinoma of the liver (FLC), in comparison with 34 cases of hepatocellular carcinoma and 8 cases of focal nodular hyperplasia. Fibrolamellar carcinoma was characterized by the presence of large amounts of tenascin in tumor stroma and by the scarce expression of basement membrane components at the contact of neoplastic clusters. Like normal hepatocytes, neoplastic cells constantly expressed the alpha1 integrin chain, lacked the beta4 integrin chain, and coexpressed E-cadherin and the hepatocyte N-related cadherin. Abnormalities in the expression of cell adhesion molecules, including altered cadherin expression, alphaV integrin chain induction, and CD44 expression, were detected in the majority of cases. The composition of the tumor stroma and the pattern of expression of cell adhesion molecules in fibrolamellar carcinoma were reminiscent of those observed in grade III and grade IV hepatocellular carcinomas. Our results therefore show that, despite its slow local growth and good prognosis, fibrolamellar carcinoma expresses many characteristics usually associated with clinically aggressive malignancies. Further studies are needed to identify the factors responsible for the apparent dissociation between clinical behavior and biological characteristics in this tumor.

Background & Aims: Cadherins and their associated molecules, such as alpha-catenin, have been shown recently to play a pivotal role in epithelial carcinogenesis., Methods: The expression of E-cadherin, N-cadherin, and alpha-catenin in 10 normal samples, 28 focal nodular hyperplasias, 9 liver cell adenomas, 65 hepatocellular carcinomas, and 9 cholangiocarcinomas was studied by immunohistochemistry and Western blotting., Results: In the normal liver, hepatocytes expressed E-cadherin and a 129-kilodalton cadherin identified by the anti-N-cadherin antibody GC4. The expression level of alpha-catenin was low. Bile duct cells expressed only E-cadherin and showed high levels of alpha-catenin. The expression of cadherins and alpha-catenin was preserved in focal nodular hyperplasia. In liver cell adenomas, cadherins and alpha-catenin were heterogeneously expressed. In hepatocellular carcinomas, cadherin and alpha-catenin expression was frequently reduced or absent. Alterations in cadherin expression correlated with large tumor size, low grade of histological differentiation, and occurrence of capsular and vascular invasion. In cholangiocarcinomas, neoplastic cells inconstantly expressed E-cadherin and alpha-catenin., Conclusions: Alterations of cadherin and alpha-catenin expression are frequent in liver cell adenomas and primary liver carcinomas. Their incidence in hepatocellular carcinomas is of prognostic significance.

Although numerous allelic chromosome losses have been reported in hepatocellular carcinomas (HCC), chromosome analysis by cytogenetic methods has rarely been performed in these tumors, unlike other solid malignant tumors. The purpose of the current study was to analyze primary liver tumors by conventional cytogenetic methods and by a new molecular cytogenetic technique, called fluorescent in situ hybridization (FISH), a technique that has been recently proposed to count the number of chromosome copies in interphase nuclei with chromosome centromeric probes. Primary cultures of tumoral cells were prepared to obtain metaphases. Specific chromosomes probes 7, 17, and 20 were used to perform in situ hybridization on isolated intact tumoral cells. Seven cases of primary liver tumors (six cases of HCC and one case of benign focal hepatic nodular hyperplasia) were investigated. A few metaphases were obtained in five of the seven tumors, and in most cases numerical abnormalities were difficult to interpret. In contrast with in situ hybridization, all cases of HCC showed losses and/or gains of chromosomes. Loss of one to three chromosomes occurred in five tumors. A gain of two chromosomes was observed in two of these five tumors. In only one case, a gain of only three chromosomes occurred. In addition, a loss of chromosome 17 was recorded for the benign tumor. These results demonstrate that FISH with specific probes can provide information on chromosome number in the tumoral cells of primary liver tumors even in the absence of analyzable metaphases. This technique opens new possibilities for the investigation of chromosome abnormalities in HCC.

Cytogenetic analysis of a new human hepatoma cell line BC2 was performed with conventional cytogenetic techniques and fluorescent in situ hybridization. Numerical and structural abnormalities were observed by conventional cytogenetics for chromosomes 1, 2, 4, 7, 8, 9, 10, 11, 15, 17 and 20. Chromosome painting allowed to specify the translocation of chromosome 1, and to characterize 3 markers from chromosome 8 and one marker from 9, which were unrecognizable by conventional techniques. Comparison of chromosome 1 abnormalities with those reported in the literature for other human hepatoma cell lines showed that structural abnormalities of chromosome 1 were present in different regions of this chromosome. A review of the literature was done, and the results discussed, suggesting that alterations of chromosome 1 may be important in hepatocarcinogenesis.

The resurgence of aldolase isozymes in cancerous tissues is a well-known but poorly understood phenomenon. This resurgence poses the problem of whether or not adult and fetal aldolase isozymes are produced by the same cells. For clarification of this question, the immunoperoxidase technique was used to locate aldolases A, B, and C in one type of fast-growing hepatoma, the LF hepatoma and, by comparison, in normal adult liver. Under optical microscopy, aldolases A and C were located in the cytoplasm of almost all of the cancerous cells. An isozyme antigenically identical with aldolase B was also demonstrated to be present in almost all of the cells, but the reaction indicating the presence of this isozyme was weaker. In normal adult liver, only aldolases A and B were demonstrated to be present in almost all the hepatocytes. Under electron microscopy in LF hepatoma, the three isozymes were found to be present mainly in the cytoplasm. These facts suggest that the three types of aldolase are very probably present in the same cells at the same time, and they provide indirect arguments leading us to think that the resurgence of fetal aldolase isozymes in cancer is not the consequence of cellular selection but is due to a disturbance at the gene control level.

The hepatoblastoma cell line HepG2 has been a matter of many investigations; most of them include biochemical studies of lipoprotein and other hepatic protein metabolism. However, the accurate cellular features of these cells have not been emphasized. We studied the cellular histologic, histochemical, and ultrastructural characteristics of this cell line. In addition, we investigated by immunoenzymatic methods the cellular biosynthesis of several proteins: apolipoproteins-AI, -B, -D, and -E, albumin, alpha-fetoprotein, transferrin, alpha-1-antitrypsin, C-reactive protein, fibronectin, and collagens I, III and IV. The rates of accumulation, in the medium of HepG2 cells, of albumin, alpha-1-antitrypsin, transferrin, and alpha-fetoprotein were 13.2 +/- 1.9; 4.9 +/- 1.5; 3.2 +/- 0.4; and 10.7 +/- 1.7 micrograms/10(6) cells/24 h, respectively. Our results show that HepG2 cells exhibited most cellular features of normal human hepatocytes. Bile canaliculi as well as Golgi apparatus complexes were particularly developed. Except for the C-reactive protein, HepG2 cells have all retained the ability to synthesize hepatic proteins but with some variable intensity from cell to cell. This hepatoblastoma cell line seems to represent a useful tool in the understanding of hepatic protein biosynthesis, particularly for the investigation on the secretory pathway of plasma proteins.

Epithelioid hemangioendotheliomas are uncommon vascular tumors, mainly observed in lung and soft tissues. Liver involvement is infrequent and, in contrast to the extrahepatic localizations of the tumor, has not been subjected to detailed ultrastructural analysis. This prompted us to report the results of the ultrastructural study of three cases of epithelioid hemangioendothelioma of the liver. Neoplastic proliferation was heterogeneous. Most of neoplastic cells presented features suggestive of endothelial differentiation, including presence of Weibel-Palade bodies and evidence of vasoformative properties demonstrated by formation of both intra- and extracellular vascular channels resembling the normal structures successively observed during embryogenesis and tissue regeneration. A minor cell population, unreported so far, exhibited ultrastructural characters resembling those of pericytes and presented organoid relationship to neoplastic endothelial cells. Tumor spreading along sinusoids induced pseudopeliotic dilatations and led to a progressive disruption of normal liver architecture. In conclusion, the three cases of hepatic epithelioid hemangioendothelioma examined in this work assume many organoid features mimicking the successive steps of normal angioformation and indicative of a high degree of morphological differentiation.

Five cases of epithelioid hemangioendothelioma of the liver are reported. This unusual type of vascular tumor is often difficult to diagnose; its angiogenic nature is not overt at the radiologic level and may be overlooked by conventional histologic examination. At imaging procedures, epithelioid hemangioendothelioma of the liver presents as multiple focal hypovascular areas, disseminated in both lobes. The combination of these imaging abnormalities with a peculiar set of demographic and clinical features, including young age, good general condition, and progressive course, is suggestive. However, the final diagnosis can be established only by histologic examination of appropriate material collected by guided liver biopsies and may be helped by immunohistochemistry and ultrastructural examination. The accurate diagnosis of epithelioid hemangioendothelioma of the liver is of clinical and therapeutic relevance. In view of its favorable clinical course, orthotopic liver transplantation may be considered for the treatment of this tumor when intrahepatic dissemination contraindicates partial hepatectomy and extrahepatic extension, excluded by exploratory laparotomy, is absent.