Altered transferrin gene expression in preneoplastic and neoplastic liver lesions induced in rats with N-nitrosomorpholine

The expression of the gene for the iron transport protein transferrin was found to be altered in preneoplastic and neoplastic lesions induced in the rat liver by N-nitrosomorpholine. The total RNA of ten hepatocellular carcinomas (HCC) was investigated by Northern blot analysis using a cDNA-probe comprising 150 bp of the 3′ region and compared with the total hepatic RNA in untreated rats. Seven hepatocellular carcinomas showed slight or pronounced reduction in transferrin expression. In situ hybridization of two additional hepatocellular carcinomas revealed marked reduction in the mRNA level for the transferrin gene compared with the surrounding tissue. In contrast, the majority of early preneoplastic lesions storing excess glycogen and tigroid cell foci expressed increased levels of transferrin mRNA. The loss of glycogen in mixed cell foci, which represent a later stage of hepatocarcinogenesis, was usually accompanied by a decrease in transferrin mRNA suggesting a close relationship between this change in gene expression and cellular dedifferentiation emerging during hepatocarcinogenesis.     

Altered transferrin gene expression in preneoplastic and neoplastic liver lesions induced in rats with N-nitrosomorpholine.

The expression of the gene for the iron transport protein transferrin was found to be altered in preneoplastic and neoplastic lesions induced in the rat liver by N-nitrosomorpholine. The total RNA of ten hepatocellular carcinomas (HCC) was investigated by Northern blot analysis using a cDNA-probe comprising 150 bp of the 3' region and compared with the total hepatic RNA in untreated rats. Seven hepatocellular carcinomas showed slight or pronounced reduction in transferrin expression. In situ hybridization of two additional hepatocellular carcinomas revealed marked reduction in the mRNA level for the transferrin gene compared with the surrounding tissue. In contrast, the majority of early preneoplastic lesions storing excess glycogen and tigroid cell foci expressed increased levels of transferrin mRNA. The loss of glycogen in mixed cell foci, which represent a later stage of hepatocarcinogenesis, was usually accompanied by a decrease in transferrin mRNA suggesting a close relationship between this change in gene expression and cellular dedifferentiation emerging during hepatocarcinogenesis.     

Fine needle aspiration biopsy of hepatocellular carcinoma and hepatocellular nodular lesions: role,controversies and approach to diagnosis

A. Wee
Fine needle aspiration biopsy of hepatocellular carcinoma and hepatocellular nodular lesions: role, controversies and appr oach to diagnosis The role of fine needle aspiration (FNA) biopsy of the liver has evolved. Advances in imaging modalities have obviated the need for tissue confirmation in clinically classic hepatocellular carcinoma (HCC). The risks of needle tract seeding and haematogenous dissemination have been actively debated. Nowadays, cytopathologists are confronted by smaller and smaller nodules, detected due to increased surveillance of high‐risk cirrhotic patients. Tissue characterization of small well‐differentiated hepatocellular nodular lesions (size less than and equal to 2 cm) is extremely challenging and has therapeutic implications. Major issues in the cytodiagnosis of HCC include: (i) distinguishing benign hepatocellular nodular lesions, namely, large regenerative nodules, dysplastic nodules, focal nodular hyperplasia and hepatocellular adenoma from reactive hepatocytes; (ii) distinguishing well‐differentiated HCC from benign hepatocellular nodular lesions; (iii) distinguishing poorly differentiated HCC from intrahepatic cholangiocarcinoma and metastatic carcinomas; (iv) determining the histogenesis of a malignant tumour; and (v) determining the site of origin of a malignant tumour. An overview of the biological evolution and histopathological aspects of dysplastic nodules, small HCCs and ‘nodule‐in‐nodule’ lesions is presented in tandem with clinically relevant nomenclature. An algorithmic approach to FNA diagnosis of HCC and hepatocellular nodular lesions is outlined. Optimal results depend on (i) a dedicated radiologist‐cytopathologist team; (ii) an on‐site cytology service, (iii) a combined cytohistological approach, (iv) immunohistochemistry, and (v) clinicopathological correlation. As we move towards personalized medicine, it is envisaged that hepatic FNA is likely to become a point of care in the management protocol as it takes on the additional role of procurement of tumour and peritumoural tissues for genomic and proteomic profiling to enable targeted molecular therapy.     

Immunohistochemical assessment of proliferating cell nuclear antigen in primary hepatocellular carcinoma and dysplastic nodules

A complementary way for the assessment of HCC prognosis is represented by the analysis of molecular markers. Thus, immunohistochemical assessment of proliferation can describe tumor aggressiveness, probability of local recurrence or metastasis potential, being very useful for the assessment of recurrence-free survival and survival until death. The aim of our study was to assess proliferating cell nuclear antigen activity in HCC and dysplastic nodules as compared with surrounding nonneoplasic areas. Immunohistochemical techniques were thus performed on the samples obtained by ultrasound-guided liver biopsies or intraoperative biopsies, in 32 patients with HCC, as well as in 3 patients with dysplastic nodules ocurring in liver cirrhosis. Expression of PCNA within extranodular areas of the HCC patients in the absence or presence of cirrhosis, was increasing from 40% to 70%, respectively. PCNA expression further increased within intranodular areas of dysplastic nodules and HCC, to 100% and 96.88%, respectively. A progressive increase of the mean values of PCNA-LI was also observed from extranodular areas without or with cirrhosis, towards intranodular areas of dysplastic nodules and HCC (4.2%, 6.8%, 31.9%, respectively). Dysplastic nodules can thus be considered lesions with a high-proliferation rate, representing an early stage of hepatocarcinogenesis. This supported the current recommendations for borderline hepatocellular nodules identified by ultrasound, which indicate an aggressive treatment similar to malignant lesions. In summary, we demonstrated a progressively increasing rate of cellular proliferation, from extranodular non-neoplasic areas to intranodular areas (dysplastic nodules and HCC), as reflected by an increased expression of proliferating cell nuclear antigen labelling index.     

Targeting stromal cells for the treatment of platelet-derived growth factor C-induced hepatocellular carcinogenesis

Non-invasive therapies for the treatment of hepatocellular carcinoma (HCC) would be of great benefit to public health. To this end, we have developed a platelet-derived growth factor-C (PDGF-C) transgenic (Tg) mouse model, which mimics many aspects of human liver carcinogenesis. Specifically, overexpression of PDGF-C results in liver fibrosis, which is preceded by activation and proliferation of hepatic stellate cells, and is followed by the development of dysplastic lesions and angiogenesis, and progression to HCCs by 8 months of age. Here, we show that PDGF-C overexpression induces the proliferation of endothelial-like cells that are present in tumors and adjacent non-neoplastic parenchyma. The protein tyrosine kinase inhibitor, imatinib (Gleevec), decreases the proliferation of non-parenchymal cells (NPC) in vitro and in vivo, with concomitant inhibition of Akt. In vivo treatment with imatinib also blocks the expression of CD34 in PDGF-C Tg mice. Decreased NPC proliferation and CD34 expression correlated with lower levels of active ERK1/2 and total levels of PDGF receptor alpha (PDGFRalpha). In summary, the small molecule inhibitor imatinib attenuates stromal cell proliferation in PDGF-C-induced HCC, which coincides with decreased expression of both CD34 and PDGFRalpha, and activated Akt. Our findings suggest that imatinib may be efficacious in the treatment of hepatocarcinogenesis, particularly when neovascularization is present.     

Lack of a relationship between immune function and chemically induced hepatocarcinogenesis in B6C3F1 mice

Summary The relationship between immune function and chemically induced hepatocarcinogenesis was studied employing an in vivo murine model. Neonatal B6C3F₁ mice were given a single carcinogenic dose of diethylnitrosamine (DEN) and the time-response kinetics for the early (foci of alteration) and late (adenomas/carcinomas) phases of hepatocellular carcinogenesis were compared to changes in hematopoiesis and immune functions associated with immune surveillance and natural resistance. Increases in hematopoiesis occurred just prior to or concurrent with the appearance of hepatocellular carcinomas, while increased macrophage and natural killer cell cytotoxicity and suppression of cell-mediated immunity occurred following tumor appearance and progressed with increasing tumor burden. Neither immunological nor hematopoietic changes were associated with early phases of hepatocarcinogenesis, as monitored by the appearance of altered hepatocellular foci. Although changes in hematopoiesis may represent an early indicator for hepatocarcinogenesis in the mouse tumor model, the data suggest that altered immune surveillance and natural resistance are not factors in the development of chemically induced hepatocellular tumors, and the changes in immune function are probably secondary to tumor development.     

Unusual histochemical pattern in preneoplastic hepatic foci characterized by hyperactivity of several enzymes

In a stop-experiment using the hepatocarcinogen N-nitrosomorpholine, as well as glycogenotic and related lesions, hepatocellular foci with a different histochemical pattern were identified. The outstanding features of these hepatic foci, which may progress to hepatocellular adenoma, were increased activities of mitochondrial glycerol-3-phosphate dehydrogenase (mG3PD), glycogen synthase, pyruvate kinase and glucose-6-phosphatase detected by enzyme histochemistry. Since no decrease in activity of any of the enzymes examined were seen in these foci, compared with normal liver, the term enzymatically hyperactive focus (EHF) is proposed for this type of lesion. Only at the stage of overtly nodular growth did these lesions exhibit some of the characteristic changes seen in nodules developing from glycogenotic foci, namely elevated activities of glucose-6-phosphate dehydrogenase, gamma-glutamyl transferase and glutathione-S-transferase P as well as decreased activities of adenosine-triphosphatase, glucose-6-phosphatase and adenylate cyclase. Some of these enzymes have been used widely in morphometric studies as markers for preneoplastic and neoplastic lesions. The inability to detect early EHF may lead to an underestimation of preneoplastic liver lesions in quantitative studies. Although there are apparent differences in the histochemical patterns of glycogen storing foci and early EHF, these differences tend to disappear during progression to overtly neoplastic lesions. In studies comparing the phenotypic alterations in different types of preneoplastic hepatic lesions, the recognition of EHF may contribute to the distinction of obligatory from facultative phenomena during transformation.     

Biochemical microanalysis of alpha-glucosidase activity in preneoplastic and neoplastic hepatic lesions induced in rats by N-nitrosomorpholine

Preneoplastic and neoplastic hepatic lesions were induced in male Sprague-Dawley rats by oral administration of N-nitrosomorpholine (NNM) for 7 weeks at a concentration of 200 mg/l of drinking-water (stop model). Using a laser dissection technique and biochemical microanalysis, the activity of the lysosomal enzyme alpha-glucosidase was measured in glycogen storage foci emerging early, and in mixed or basophilic cell populations (foci and carcinomas) appearing later during hepatocarcinogenesis. In the liver tissue of normal appearance in both untreated controls and NNM-treated animals a slight gradient of alpha-glucosidase activity was observed leading from relatively high activities in zone 1 to lower activities in zone 3 of the liver lobule. In preneoplastic glycogen storage foci a considerable relative reduction in alpha-glucosidase activity was detected, suggesting that a decrease in the hydrolytic glycogen degradation contributes to the disturbance in phosphorylytic glycogen breakdown observed earlier in the majority of the glycogenotic foci. In contrast with glycogen storage foci, mixed and basophilic cell foci and particularly hepatocellular carcinomas showed a marked increase in alpha-glucosidase activity compared with that of normal liver tissue. The gradual enhancement in enzyme activity appeared to be closely related to the reduction in glycogen initially stored in excess during the later stages of hepatocarcinogenesis. The results support the concept that a fundamental shift in carbohydrate metabolism is characteristic of neoplastic transformation of hepatocytes.     

Premalignant Variations in Extracellular Matrix Composition in Chemically Induced Hepatocellular Carcinoma in Rats

Chemical composition of extracellular matrix (ECM) plays a pivotal role in cellular and tissue development, regeneration, and differentiation. It also plays a key role in pathogenesis of hepatocellular carcinoma (HCC). This study explored premalignant changes in the liver tissue content of collagen (as hydroxyproline, HP), total glycosaminoglycans (TGAGs), free glucosamine (FGA), total sialic acid (TSA), lysosomal membrane integrity variations (calculated as total and free cathepsin D activities), and liver histology. Serum alfa-fetoprotein (AFP) level was used as an early marker for HCC in two groups of Wistar rats. One group of rats served as control and was provided normal saline orally. The other group was provided trichloroacetic acid (TCA) as 0.5 g/kg/day for five consecutive days by oral gavage. Animals were killed before tumor development. The treatment revealed dysplastic changes in addition to microsteatosis (fatty changes). Both sinusoids and the portal vein among dysplastic cells were dilated and congested. These dysplastic foci are believed to be premalignant and may be precancerous lesions. The following things were observed: a highly significant increase in serum AFP (as a key marker for HCC), a significant decrease in HP and TSA, a significant increase in FGA, nonsignificant decrease in TGAGs, significant up-regulation of free cathepsin D, nonsignificant decrease in total cathepsin D activities, and destabilization of lysosomal membrane integrity. Down-regulation of HP, TSA, and TGAGs seems to be a prerequisite for cancer development. This might be stimulated by up-regulation of free cathepsin D activity. Perhaps tissue fibrosis is not a condition for developing HCC because collagen was significantly depressed. Up-regulated FGA could be assumed to be a defense mechanism against TCA-induced proteolysis of membrane proteins because it is frequently reported to be of value in cancer chemotherapy. Studied ECM perturbations can be assumed as preliminary changes during chemical hepatocarcinogenesis at the tissue level. Prospective studies on blood levels of cathepsins, TGAGs, and individual ECM variables such as TSA, FGA, and Hp in patients at risk for HCC, performed in parallel with assessments of AFP, may provide a cost-effective way to find new links between tissue changes and circulation that would permit early prediction of disease. It may also provide a way to monitor HCC and compensate for the missed peak AFP values.     

The Role of Indoleamine 2,3-Dioxygenase in Diethylnitrosamine-Induced Liver Carcinogenesis

Indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing intracellular enzyme of the L-kynurenine pathway, causes preneoplastic cells and tumor cells to escape the immune system by inducing immune tolerance; this mechanism might be associated with the development and progression of human malignancies. In the present study, we investigated the role of IDO in diethylnitrosamine (DEN)-induced hepatocarcinogenesis by using IDO-knockout (KO) mice. To induce hepatocellular carcinoma (HCC), hepatic adenoma, and preneoplastic hepatocellular lesions termed foci of cellular alteration (FCA), male IDO-wild-type (WT) and IDO-KO mice with a C57BL/6J background received a single intraperitoneal injection of DEN at 2 weeks of age. The mice were sacrificed to evaluate the development of FCA and hepatocellular neoplasms. HCC overexpressed IDO and L-kynurenine compared to surrounding normal tissue in the DEN-treated IDO-WT mice. The number and cell proliferative activity of FCAs, and the incidence and multiplicity of HCC were significantly greater in the IDO-WT than in the IDO-KO mice. The expression levels of the IDO protein, of L-kynurenine, and of IFN-γ, COX-2, TNF-α, and Foxp3 mRNA were also significantly increased in the DEN-induced hepatic tumors that developed in the IDO-WT mice. The mRNA expression levels of CD8, perforin and granzyme B were markedly increased in hepatic tumors developed in IDO-KO mice. Moreover, Foxp3-positive inflammatory cells had infiltrated into the livers of DEN-treated IDO-WT mice, whereas fewer cells had infiltrated into the livers of IDO-KO mice. Induction of IDO and elevation of L-kynurenine might play a critical role in both the early and late phase of liver carcinogenesis. Our findings suggest that inhibition of IDO might offer a promising strategy for the prevention of liver cancer.     

Mechanisms of human hepatocarcinogenesis

The major risk factors and etiological agents responsible for development of hepatocellular carcinoma in humans have been identified and characterized. Among these are chronic infection with hepatitis B virus or hepatitis C virus, exposure to aflatoxin B1, and cirrhosis of any etiology (including alcoholic cirrhosis and cirrhosis associated with genetic liver diseases). Both chronic hepatitis and cirrhosis represent major preneoplastic conditions of the liver as the majority of hepatocellular carcinomas arise in these pathological settings. Hepatocarcinogenesis represents a linear and progressive process in which successively more aberrant monoclonal populations of hepatocytes evolve. Regenerative hepatocytes in focal lesions in the inflamed liver (chronic hepatitis or cirrhosis) give rise to hyperplastic hepatocyte nodules, and these progress to dysplastic nodules, which are thought to be the direct precursor of hepatocellular carcinoma. In most cases, the neoplastic transformation of hepatocytes results from accumulation of genetic damage during the repetitive cellular proliferation that occurs in the injured liver in response to paracrine growth factor and cytokine stimulation. Hepatocellular carcinomas exhibit numerous genetic abnormalities (including chromosomal deletions, rearrangements, aneuploidy, gene amplifications, and mutations), as well as epigenetic alterations (including modulation of DNA methylation). These genetic and epigenetic alterations combine to activate positive mediators of cellular proliferation (including cellular proto-oncogenes and their mitogenic signaling pathways) and inactivate negative mediators of cellular proliferation (including tumor suppressor genes), resulting in cells with autonomous growth potential. However, hepatocellular carcinomas exhibit a high degree of genetic heterogeneity, suggesting that multiple molecular pathways may be involved in the genesis of subsets of hepatocellular neoplasms. Continued investigation of the mechanisms of hepatocarcinogenesis will refine our current understanding of the molecular and cellular basis for neoplastic transformation in liver, enabling the development of effective strategies for prevention and/or more effective treatment of hepatocellular carcinoma.     

Expression of inducible nitric oxide (NO) synthase but not prevention by its gene ablation of hepatocarcinogenesis with fibrosis caused by a choline-deficient, L-amino acid-defined diet in rats and mice.

Expression of inducible nitric oxide synthase (iNOS) and effects of iNOS gene ablation on the hepatocarcinogenesis associated with fibrosis caused by a choline-deficient, L-amino acid-defined (CDAA) diet, were examined in male F344 rats and C57BL/6J wild-type and iNOS-/- mice. Western blot, RT-PCR and immunohistochemical analyses revealed increased expression of iNOS protein and mRNA in the livers of rats and wild-type mice fed a CDAA diet for 12-80 weeks, associated with elevated serum NO(x) and liver nitrotyrosine levels. iNOS-/- mice demonstrated greater liver injury and fibrosis in the early stage than their wild-type counterparts, but this did not significantly affect the incidence and multiplicity of altered foci, adenomas and hepatocellular carcinomas in spite of immunohistochemical iNOS expression in these lesions. Results suggested no major determinant roles of the expressed iNOS in the development of liver tumors caused by the CDAA diet.     

Decreased density of beta1-adrenergic receptors in preneoplastic and neoplastic liver lesions of F344 rats

There is some evidence that rodent hepatocarcinogenesis is accompanied by changes in the adrenergic responsiveness of liver cells to catecholamines. In this study, immunohistochemical expression of beta1-adrenergic receptors (beta1-ARs) has been examined in spontaneous and chemically induced preneoplastic and neoplastic liver lesions of female and male Fischer 344 rats. An antibody specific for beta1-AR subtype was used. The study was carried out on archival formalin-fixed and paraffin-embedded livers from rats used in a previous study of hepatocarcinogenesis. One control group given distilled water by gavage, and two experimental groups, one initiated with a single dose of diethylnitrosamine (DEN) and one initiated with DEN and continuously treated with phenobarbital (PB) were examined. Rats were sacrificed after 2, 4, 8 and 21 months of experimentation. All types of liver putative preneoplastic lesions examined (basophilic, glycogen-retaining, or mixed cell foci) show a lower density of beta1-ARs than the surrounding normal liver parenchyma, either in control and in DEN-treated or DEN+PB-treated rats. No immunostaining is detectable in several altered cell foci. Hepatocellular adenomas and hepatocellular carcinomas also show a very low density of beta1-ARs, extensive areas completely devoid of beta1-ARs being mingled with areas showing a weak immunostaining.     

Cellular origin of hepatocellular carcinomas

There are four levels of cells in the hepatic lineage which may respond to different carcinogenic regimens: (1) the mature hepatocyte, which responds to diethylnitrosamine (DEN) hepatocarcinogenesis. (2) The bile duct progenitor cells, which give rise to cholangiocellular carcinomas when the furan model is used or when hamsters infected with liver flukes (Clornorchis sinensis) are exposed to dimethylnitrosamine. (3) The ductular 'bipolar' progenitor cell which gives rise to hepatocellular carcinomas (HCC) in several N-2-acetylaminofluorene (N-2-AAF) based regimens, and (4) the periductular stem cell, which is the cell of origin of HCC induced by the choline deficiency models of hepatocarcinogenesis. Extrahepatic (bone marrow) origin of the periductular stem cells is supported by recent data showing that hepatocytes may express genetic markers of donor hematopoietic cells after bone marrow transplantation.     

Highly well differentiated hepatocellular carcinoma and benign hepatocellular lesions. Can they be distinguished on fine needle aspiration biopsy?

OBJECTIVE: To determine whether highly well differentiated hepatocellular carcinoma can be distinguished from benign hepatocellular lesions on fine needle aspiration biopsy (FNAB). STUDY DESIGN: Ninety-five FNABs from 88 patients with hepatic masses/diffuse conditions were reviewed according to new cytologic criteria established by Takenaka et al. They were classified into well-, moderately and poorly differentiated hepatocellular carcinomas (W-, M- and P-HCC) and benign aspirates and histologically verified. RESULTS: There were 21 W-HCC, 39 M-HCC, 10 P-HCC, 3 problematic and 22 benign aspirates. The most useful criteria for diagnosing highly W-HCC were architectural features on the smears/cell block sections, including hypercellularity; arborescent, cohesive clusters; broad trabeculae; transgressing and peripheral endothelium; and cytologic details of small, monotonous hepatocytes with nuclear crowding, decreased cytoplasm, increased nuclear/cytoplasmic ratio, atypical naked nuclei and tumor giant cells. Well-defined cytoplasmic borders, abundant thick and monotonous cytoplasm, eccentric nuclei, thick nuclear membranes, irregular nuclear contours, increased chromatin density, irregular chromatin distribution and macronucleoli were not always detectable in highly W-HCC. In fact, some of them were seen in dysplastic hepatocytes. Deficient reticulin patterns and diffuse sinusoidal CD34 reactivity were helpful. CONCLUSION: Experience, attention to architectural and cytologic details in smears/cell blocks and clinicopathologic correlation should reduce the number of indeterminate reports. However, there will always remain some cytohistologically challenging cases.     

Morphometric characteristics of hepatocellular dysplasia

Morphometric study of liver biopsies from six entities (normal tissue, post-hepatitis cirrhosis, post-alcoholic cirrhosis, cancer-related cirrhosis, hepatocellular adenoma and hepatocellular adenocarcinoma) confirmed that this technique can be a valuable adjunct to histopathologic study in the examination of such specimens. As expected, measurements in cirrhotic nodules showed two populations of cells. The so-called "large dysplastic cells" had nuclear and cellular areas close to those of normal hepatocytes and should thus be considered to be hyperplastic elements, not precancerous elements. The smaller dysplastic cells had morphometric values close to those of the corresponding hepatocellular carcinomas, indicating that these cells are the truly precancerous ones. Therefore, while the study confirmed that hepatic cirrhosis is a precancerous lesion, it also showed that the term hepatocellular dysplasia must be restricted to the smaller type of cells found in such nodules.