Lamellar fibrosis in the fibrolamellar variant of hepatocellular carcinoma: a role for transforming growth factor beta.

Transforming growth factor-beta (TGF-beta) is a pluripotent regulatory molecule, found in at least five different isoforms. It is produced in many different organs. In the liver, TGF-beta is expressed in non-parenchymal cells, but not in hepatocytes. This growth factor is known to induce fibrosis in the course of a variety of pathologic processes. Recently, TGF-beta has also been identified in hepatocellular carcinoma (HCC) cells, and the suggestion has been made that this growth factor may play a role in hepatocarcinogenesis. In this study, we report the findings of immunohistochemical stains for TGF-beta, performed on paraffin sections of 14 human HCCs of the usual type and 11 examples of the fibrolamellar variant (FLC). TGF-beta was detected in tumor cells of 3 HCCs (21%) and 9 FLCs (82%). Compared with the HCCs, the FLCs displayed a more diffuse and intense staining pattern for TGF-beta. Our findings suggest that lamellar fibrosis, which is a histologic hallmark of FLC, may be due to the action of TGF-beta produced by tumor cells.

Large-cell change of hepatocytes in cirrhosis may represent a reaction to prolonged cholestasis.

Large-cell change of hepatocytes (LCC), also called liver cell dysplasia of large-cell type, is a set of cytologic changes comprising nuclear and cytoplasmic enlargement, nuclear pleomorphism, and multinucleation. This entity is encountered frequently on histologic or cytologic examination of specimens obtained from livers with a variety of chronic diseases and originally was thought to have a premalignant nature. Accumulating evidence, however, now suggests that LCC is merely a reactive change. Having often observed LCC in liver specimens with chronic biliary tract disease, that is, in livers where cholestasis preceded hepatocyte injury, we surmised that LCC may be a result of prolonged cholestasis. To determine whether there was any association between LCC and cholestasis, we examined microscopically a series of 400 nodules from 40 consecutive adult cirrhotic livers, resected on transplantation, and graded LCC and cholestasis semiquantitatively. LCC was present diffusely in cirrhotic nodules of 25 specimens (62.5%). Nine additional specimens (22.5%) had focal mild LCC. Usually, LCC and cholestasis occurred together, in the same cirrhotic nodules and in the same areas of nodules. There was a statistically significant association between the presence and grade of LCC and those of cholestasis (p < 0.0001; chi-square test). Within etiological categories of cirrhosis (chronic hepatitis; n = 28; alcoholic liver disease; n = 6; biliary disease: n = 6), the significance was maintained. We conclude that, in cirrhosis of different etiologies, LCC may represent a reactive change that results from prolonged cytoplasmic cholestasis.

Alcoholism is associated with hepatitis C but not hepatitis B in an urban population.

OBJECTIVES: Previous studies have suggested an association of viral hepatitis with alcoholism, although the role of confounding risk factors (e.g. i.v. drug use) has not been adequately excluded. We therefore compared the seroprevalences of hepatitis B and C in alcoholic patients to that of a nonalcoholic control group. METHODS: Hepatitis B surface antigen, hepatitis B core antibody, hepatitis B surface antibody, and hepatitis C virus antibody testing (second generation ELISA and a confirmatory recombinant immunoblot assay) was performed in 150 consecutive alcoholics admitted for detoxification and in 166 randomly selected patients attending a general medical clinic who were screened for alcoholism. RESULTS: Hepatitis B and C seropositivities in actively drinking alcoholics are 49.3 and 35.3%, respectively, and were significantly associated with a history of i.v. drug abuse. Out of 166 general medicine clinics patients, 93 were classified as nonalcoholic (by both self-report and collateral verification), 46 patients had a history of alcoholism , and 27 were indeterminate. In the subgroup of patients without known viral hepatitis risk factors, there was no significant difference in hepatitis B seropositivity among nonalcoholic general medicine clinic patients, alcoholic general medicine clinic patients, and alcoholic patients admitted for detoxification (22.1%, 30.3%, and 27.6%, respectively). In contrast, anti-HCV recombinant immunoblot assay seropositivity in alcohol patients admitted for detoxification without risk factors was significantly greater than in nonalcoholic general medicine patients without risk factors (10 vs 0%, p >0.01). Stepwise logistic regression analysis revealed that alcoholism requiring detoxification was a significant risk factor for hepatitis C but not for hepatitis B seropositivity. CONCLUSIONS: The increased seroprevalence of hepatitis C in actively drinking alcoholic patients without known risk factors suggests that alcoholism, in some way, is a predisposing factor for HCV infection.

DNA image cytometric analysis of primary clear cell carcinoma of the liver.

Eleven cases of clear cell hepatocellular carcinoma were evaluated for DNA ploidy by means of image analysis of Feulgen-stained tissue sections. The tumors displayed a trabecular or solid pattern and contained between 40% and 90% clear cells. Five tumors (45.4%) were diploid and six tumors (54.5%) were nondiploid (four aneuploid, one tetraploid, and one multiploid). The diploid tumors had no significant pleomorphism or mitotic activity, whereas the nondiploid tumors displayed a moderate to severe degree of pleomorphism and high mitotic rate. In addition, the nuclear area of the aneuploid tumor cells (55.3 microns2 +/- 5.4%, mean +/- SD) was significantly larger than that of the control hepatocytes (43.3 microns2 +/- 6.8%) (P = .01) and that of the diploid tumor cells (39.5 microns2 +/- 5.5%) (P = .001). We conclude that clear cell carcinoma of the liver comprises two groups: one with bland morphologic features and diploid DNA content and the other with aggressive morphologic features and aneuploid DNA content. We suggest that this finding may explain the discrepant literature reports regarding the prognosis of this tumor.

DNA ploidy of fibrolamellar hepatocellular carcinoma by image analysis.

Twelve cases of fibrolamellar hepatocellular carcinoma (FLC) were evaluated for DNA ploidy by means of image analysis of Feulgen-stained tissue sections. All of the tumors showed a nondiploid DNA distribution (six aneuploid and six tetraploid); no diploid pattern was found. The nuclear area of the tumors (53.8 microns 2 +/- 18.0; mean +/- standard deviation) was significantly larger than that of the surrounding noncancerous livers (33.2 +/- 4.7; P < .0001). These findings suggest that DNA content in fibrolamellar carcinoma (FLC) is not directly related to the clinical behavior and that other factors may be responsible for the better prognosis of this variant of HCC.

DNA ploidy analysis of hepatic preneoplastic and neoplastic lesions in woodchucks experimentally infected with woodchuck hepatitis virus.

We analyzed the DNA ploidy and the nuclear size of hepatocytes within hepatocellular carcinoma, putative preneoplastic (clear cell and basophilic foci) and adjacent non-neoplastic liver in 30 woodchucks neonatally infected with the woodchuck hepatitis virus. In livers from control woodchucks, in clear cell foci and in most chronic portal hepatitis, the hepatocytes were diploid, with less than 10% tetraploid cells. Aneuploid peaks were found in 50% of the livers with chronic active hepatitis, in 63% of basophilic foci and in 90% of hepatocellular carcinoma. Within the same tumor, aneuploid peaks with different DNA indices were observed frequently, indicating heterogeneity of tumor. S-phase was always elevated, indicating an increased rate of proliferation. Aneuploid cells had nuclei that were larger than those of control liver cells. In some basophilic foci and in some livers with chronic active hepatitis, abnormal DNA was demonstrated before the development of hepatocellular carcinoma, suggesting that these may be populations of hepatocytes at risk of neoplastic transformation.

Progressive impairment of monocytic function in HIV-1-infected human macrophage hybridomas.

Using human macrophage hybridomas infected with HIV-1, we investigated monocyte function over a 5-week period after HIV-1 infection. Two clones, 63 and 30, were infected with HIV-1IIIB. Infection was documented by RT activity (15 x 10(6) cpm/ml), intracytoplasmic staining with an anti-p24 antibody, in situ hybridization with an HIV-1-specific riboprobe, and electron microscopy showing intracytoplasmic virus. Two weeks after infection, clones 63 and 30 lost expression of all class II antigens (DR, 81.7 vs. 0%; DQ, 15.6 vs. 0%; and DP, 76.9 vs. 0%) while retaining expression of class I (87.4 vs. 84.1%), LFA-1 (82.4 vs. 83.1%), and LFA-3 (79.1 vs. 74.7%) antigens when compared to uninfected cells. When tested for functional integrity, infected but not uninfected clone 63 cells failed to stimulate a tetanus-specific MHC-restricted T cell proliferative response 2 weeks after infection. Cytokine secretion and antigen processing were also perturbed as production of IL-1 was abolished 2 weeks after infection (although IL-6 secretion was augmented) and infected clone 63 cells failed to process exogenous antigen. Last, the viability of T cells cocultured with infected clone 63 was dramatically decreased 35 days after infection (85 vs. 15%). There was no evidence of transmission of HIV-1 to T cells, suggesting a toxic effect of infected clone 63. Taken together, these data suggest that altered macrophage function in our system occurs at multiple levels, which may account for the early immunological defects described in HIV-1 infection.

Image analysis versus flow cytometry for DNA ploidy quantitation of solid tumors: a comparison of six methods of sample preparation.

With the availability of user-friendly interactive image analysis instruments for DNA analysis, there is a growing need for comparison with the established methodology of flow cytometry. We have compared the results of DNA ploidy quantitation in 12 solid tumors prepared by six different techniques of sample preparation: flow cytometry of fresh cell suspensions and of nuclei isolated from formalin-fixed, paraffin-embedded tissue; and image analysis of touch preparations, of disaggregated cells from paraffin-embedded tissue as well as of 3- and 7-microns-thick tissue sections. Complete agreement in DNA ploidy results obtained by the six methods was found in six out of 12 solid tumors. Image analysis of touch preparations detected most tetraploid and multiple aneuploid peaks. Sections of 7-microns-thick tissue gave better histogram quality than 3-microns-thick sections, however tetraploid peaks were not resolved in one case. Image analysis of disaggregated paraffin-embedded tumor showed comparable ploidy to fresh touch preparations in seven out of 12 cases, the discrepancies being due to loss of tetraploid or multiple aneuploid peaks. Flow cytometry gave good histograms, but tetraploid and multiple aneuploid peaks were occasionally not detected. Each method presents advantages and disadvantages. Flow cytometry and image analysis are complementary methods for DNA quantitation, and more than one method may be necessary to confirm the DNA content of solid tumors.

DNA image cytometric analysis of macroregenerative nodules (adenomatous hyperplasia) of the liver: evidence in support of their preneoplastic nature.

Twenty-eight macroregenerative nodules from 14 cirrhotic patients who underwent orthotopic liver transplantation were evaluated for DNA ploidy by means of image analysis of Feulgen-stained tissue sections. The lesions were classified as type 1 (16 cases) or type 2 (12 cases) on the basis of the absence or presence of cellular or architectural atypia in the nodules. The surrounding cirrhotic nodules were evaluated for liver cell dysplasia. Aneuploid peaks were significantly more frequent in type 2 macroregenerative nodules (58.3%) than in the cirrhotic regenerative nodules (7.1%) (p < 0.007). In addition, aneuploid peaks occurred with increased frequency in type 2 nodules (58.3%) than in type 1 macroregenerative nodules (6.2%) (p < 0.02). Only two aneuploid peaks (14.2%) were found in dysplastic cirrhotic livers. The nuclear area of aneuploid hepatocytes (71.6 microns 2 +/- 10.1%, mean +/- S.D.) differed significantly from that of diploid liver cells (45.4 microns 2 +/- 6.5%) (p < 0.0001). Tetraploid peaks occurred in three type 2 lesions (25%); they were also found in one type 1 macroregenerative nodule (6.2%), one cirrhotic liver without dysplasia (7.1%) and three cirrhotic livers with dysplasia (21.4%). These findings support the notion that macroregenerative type 2 nodules are directly implicated in hepatocarcinogenesis and that their presence should be sought as an indicator of malignant potential in cirrhotic livers.

Hepatitis C virus antibody in alcoholic patients. Association with the presence of portal and/or lobular hepatitis.

OBJECTIVE: To evaluate the relationship between hepatitis C viral infection and alcoholic liver disease. DESIGN: Case-comparison study. SETTING: Bronx (NY) Veterans Affairs Medical Center. PARTICIPANTS: Forty-seven consecutive alcoholic patients undergoing diagnostic liver biopsy. MAIN OUTCOME MEASURES: Serum was obtained at the time of liver biopsy and assayed for antibodies to hepatitis C virus using enzyme-linked immunosorbent assay, recombinant immunoblot assay, and hepatitis C virus neutralization methods. RESULTS: Antibody to hepatitis C virus, as confirmed by the recombinant immunoblot assay, was strongly associated with the presence of portal and/or lobular inflammation (91% seropositivity) but was only present in 16% of patients without this histologic finding (P < .001). In patients without portal or lobular hepatitis, recombinant immunoblot assay seropositivity was seen in 27% of patients with cirrhosis and 20% of patients with alcoholic hepatitis and was absent in patients with steatosis and/or perivenular fibrosis. In the subgroup of alcoholic patients who were without known risk factors for hepatitis C virus infection (ie, no history of intravenous drug use or blood transfusions), antibody to hepatitis C virus was present in 78% of subjects with portal and/or lobular hepatitis but was absent in those with other types of alcoholic liver disease. Finally, anti-hepatitis C virus-seropositive patients had a significantly greater mean necroinflammatory score as compared with anti-hepatitis C virus-seronegative alcoholic patients (2.1 vs 1.2; P < .001). In contrast, there was no significant difference in the mean fibrosis score between the two groups. CONCLUSIONS: The presence of portal and/or lobular inflammation is strongly associated with antibodies to hepatitis C virus in alcoholic patients, even in the absence of known risk factors. This association indicates that hepatitis C virus is responsible, at least in part, for the portal and/or lobular hepatitis associated with alcoholic liver disease.

Immunoelectron microscopy identification of early proliferating cells in rat liver tissue during hyperplasia induced by lead nitrate.

Recent studies have suggested that hepatic stem cells may be involved in at least some forms of liver epithelial growth. To obtain further information on this controversial hypothesis, we treated rats with lead nitrate to induce liver growth and identified the cells undergoing early DNA synthesis by bromodeoxyuridine immunohistochemistry, using both light and electron microscopic detection methods. Eight hours after an intravenous injection of lead nitrate 100 mumol/kg, DNA synthesis was detected in a few scattered hepatocytes and in nonparenchymal cells in portal connective tissue. At the light microscopic level, identification of nonparenchymal cells was limited to bile duct epithelial cells. Other cell types were also labeled, but their identity could not be established. At the ultrastructural level, however, four types of nonparenchymal cells were identified as containing bromodeoxyuridine immunogold particles. These four types included bile duct epithelial cells, fibroblasts, macrophages and nondescript periductular cells. These periductular cells displayed certain ultrastructural features of bile duct cells but did not line a lumen or display microvilli on their apical membrane, nor did they reside within the bile duct basement membrane. Because proliferation of nonparenchymal cells in portal areas preceded that of hepatocytes, it is suggested that the former reaction reflects a direct mitogenic effect of lead nitrate and not an adaptive growth response secondary to parenchymal enlargement. However, whether DNA synthesis in periductular cells or bile duct cells reflects activation of hepatic stem cells cannot be established from the present morphological observations. If so, such a progenitor compartment must be dormant because it does not seem to play a functional role in this and other forms of adult liver epithelial growth.

Productive nonlytic human immunodeficiency virus type 1 replication in a newly established human leukemia cell line.

We have isolated a lymphoid cell line, MDS, from the pleural exudate of a patient with chronic myelomonocytic leukemia. The cells are biphenotypic, containing various T-cell and myeloid markers, and are surface negative for CD4 and CD8 but have low CD4 mRNA. The cells grow in suspension with a doubling time of 15 hr, have been karyotyped as trisomy 21, are negative for human immunodeficiency virus type 1 (HIV-1), and are tumorigenic in the nude mouse. We have isolated two stable HIV-1-producing cell lines, MDS-T, by transfecting MDS cells with pHXBc2, and MDS-I, by infecting MDS cells with HIV-1IIIB. In 24 hr, 1 x 10(5) MDS-T or MDS-I cells produce 46 ng of p24 per ml and reverse transcriptase that is capable of incorporating 0.2 pmol of [32P]TTP into oligo(dT).poly(A). Ultrastructural studies showed numerous mature viral particles in MDS-T and MDS-I cells that are capable of infecting T cells. HIV-1 infection could be inhibited by 25% in the MDS cells with the anti-CD4 antibody Leu 3a. For over a year MDS-T and MDS-I cells have been producing high concentrations of HIV-1 in culture. A subclone derived from the MDS cells behaves like the parent cells when transfected or infected with HIV-1. In contrast to other T-cell lines, neither phorbol 12-myristate 13-acetate nor tumor necrosis factor alpha stimulated the replication of HIV-1, whereas bromoadenosine 3',5'-cyclic monophosphate or interferon alpha caused 50% and 80% inhibition of reverse transcriptase production, respectively. These chronically infected T-cell lines are a useful model system to study the effect of anti-HIV agents and cellular factors required for HIV-1 replication.

Expression of sialylated Lewis(x) antigen in chronic and neoplastic liver diseases.

Phenotypic expression of sialylated Lewis(x) antigen by means of the monoclonal antiserum SNH3 was studied in 87 livers, which included normal and steatotic livers and livers with chronic persistent and chronic active hepatitis, alcoholic hepatitis, allograft rejection, focal nodular hyperplasia, hepatocellular carcinoma, cholangiocarcinoma, metastatic carcinoma, cirrhosis of various causes (autoimmune, alcoholic, viral, drug induced, Wilson's disease, and primary biliary cirrhosis). The biotin-streptavidin-peroxidase method was used on formaldehyde-fixed, paraffin-embedded sections. Sialylated Lewis(x) antigen was not demonstrated in normal livers. Hepatocellular expression in a diffuse or perinodular honeycomb pattern was seen in cirrhosis, irrespective of cause. Sialylated Lewis(x) antigen was also observed in hepatocytes around metastatic carcinoma in the absence of inflammation, cirrhosis, or regeneration. Some bile ductules, most likely ductular hepatocytes, but not bile ducts, expressed sialylated Lewis(x) antigen. Sialylated Lewis(x) antigen was seen diffusely in fibrolamellar hepatocellular carcinoma, focally in other hepatocellular carcinomas, and either focally or diffusely in cholangiocarcinomas.

Histogenesis of bile duct-like cells proliferating during ethionine hepatocarcinogenesis. Evidence for a biliary epithelial nature of oval cells.

The origin of bile duct-like cells (oval cells) proliferating during chemical hepatocarcinogenesis is highly controversial. To illuminate this issue, we induced oval cell proliferation by feeding rats a choline-devoid diet containing 0.1% ethionine (CDE), a hepatocarcinogenic diet, for up to 60 days. At various times we studied 1) oval cell morphology by light and electron microscopy, 2) the immunohistochemical expression of albumin and intermediate filament proteins by the various hepatic cells, 3) hepatic incorporation of [3H]thymidine by histoautoradiography, 4) the fractional area occupied by duct-like structures in liver cross sections, 5) the biliary tree volume in vivo to establish the possible continuity of the proliferated structures to the existing biliary lumina, and 6) spontaneous bile flow rate and the choleretic responsiveness to the hormone secretin, which stimulates ductular secretory activity. The results demonstrated the following: 1) oval cells resemble bile duct cells with respect to their histologic and ultrastructural appearance and their formation of duct-like structures; 2) as normal and hyperplastic bile duct cells induced by bile duct ligation, oval cells are positive for cytokeratins 7 and 19 (markers of glandular epithelia) and 8 and 18 (markers of simple epithelia) and are negative for vimentin and desmin, markers of mesenchymal and muscular differentiation, respectively; 3) in general, oval cells are negative for albumin, which is expressive of hepatocyte lineage, even though a few are positive for this protein, particularly those morphologically resembling small hepatocytes; 4) after initiation of the CDE diet, DNA synthesis begins in biliary epithelial cells; and 5) the degree of oval cell proliferation parallels the increase in biliary tree volume, spontaneous bile flow rate, and responsiveness to secretin choleresis, as in bile duct cell hyperplasia induced by biliary obstruction. Although the involvement of a periductular progenitor compartment cannot entirely be eliminated, these findings are construed to indicate that oval cells proliferating during CDE hepatocarcinogenesis are biliary epithelial cells. In our view, oval cells represent the two-dimensional expression of spatially expanded cholangioles and intrahepatic bile ductules and/or ducts.

Severity of Helicobacter-induced gastric injury correlates with gastric juice ammonia.

We postulated that ammonia produced by Helicobacter pylori may contribute to gastric mucosal injury. This hypothesis was evaluated in Helicobacter-positive patients with chronic renal failure in whom a high urea concentration might amplify this phenomenon. Gastric urea and ammonia were measured, and the severity of gastritis was evaluated by counting mononuclear and polymorphonuclear cells. High gastric ammonia and low urea in Helicobacter-positive patients, and the converse in Helicobacter-negative subjects, were observed. There was a significant correlation between gastric ammonia and interstitial polymorphonuclear leukocytes infiltration (P less than 0.05), suggesting a causal link. Eradication of Helicobacter pylori was associated with a decrease of ammonia and an increase of urea (P less than 0.01). The significant correlation between the severity of gastric inflammation and the gastric juice ammonia concentration suggests that ammonia may play a pathogenic role in Helicobacter-associated gastric injury.

Chronic alcoholic gastritis. Roles of alcohol and Helicobacter pylori.

We assessed the relative roles of alcohol and infection with Helicobacter pylori in the pathogenesis of chronic gastritis in alcoholic patients. Helicobacter pylori was found in 14 of 18 alcoholics with dyspepsia and was associated with chronic antral gastritis. Gastric biopsy specimens were normal in four H pylori-negative alcoholics. Studies were repeated 3 to 4 weeks after controlled abstinence. There was no change in histologic findings during this period, indicating that alcohol itself was not the major causative agent. We then eliminated H pylori in 10 subjects by giving triple therapy (bismuth subsalicylate, amoxicillin, and metronidazole). Treatment for H pylori was associated with almost complete normalization of histologic findings. Four control subjects who received antacids alone showed no improvement. Dyspeptic symptoms in H pylori-positive patients significantly improved after elimination of this organism, whereas there was no change with antacid treatment.

A nude mouse model for the in vivo production of hepatitis B virus.

Hepatitis B virus genome-transfected HepG2 cells (2.2.15 cells) inoculated into nude mice produced tumors within 2-8 wk. Dane particles, hepatitis B virus deoxyribonucleic acid polymerase activity, hepatitis B surface antigen, and hepatitis B e antigen were detected in the serum, and 36% of mice developed antibodies to hepatitis B core antigen. In the tumors, hepatitis B surface, core, and e antigens were observed by electron microscopy and immunoenzymatic techniques. In-situ hybridization and Southern blot analysis showed hepatitis B virus deoxyribonucleic acid in the tumor. Tumors could be propagated by injection of minced tumor tissue or of a tumor-derived cell line. Liver of tumor-bearing mice as well as sera and tissues of mice inoculated with control cell lines did not show hepatitis B virus genome or viral markers. Tumors induced by both 2.2.15 and nontransfected HepG2 cells exhibited myc oncogene protein and various hepatoma-associated antigens (alpha-fetoprotein, alpha-1-antitrypsin, alpha-1-antichymotrypsin, carcinoembryonic antigen, cytokeratin), suggesting that viral formation does not interfere with expression of these antigens. This experimental model will be helpful to study the effect of drugs on in-vivo hepatitis B virus replication and viral antigen expression.