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Qidong City, China, has had high liver cancer incidence from endemic hepatitis B virus (HBV) infection and dietary exposure to aflatoxin. Based on etiologic studies, we began interventions in 1980 to reduce dietary aflatoxin and initiate neonatal HBV vaccination. We studied trends in liver cancer incidence rates in the 1.1 million inhabitants of Qidong and examined trends in aflatoxin exposure, staple food consumption, HBV infection markers and annual income. Aflatoxin exposure declined greatly in association with economic reform, increased earnings and educational programs to shift staple food consumption in the total population from moldy corn to fresh rice. A controlled neonatal HBV vaccination trial began in 1983 and ended in November, 1990, when vaccination was expanded to all newborns. Liver cancer incidence fell dramatically in young adults. Compared with 1980–83, the age-specific liver cancer incidence rates in 2005–08 significantly decreased 14-fold at ages 20–24, 9-fold at ages 25–29, 4-fold at ages 30–34, 1.5-fold at ages 35–39, 1.2-fold at ages 40–44 and 1.4-fold at ages 45–49, but increased at older ages. The 14-fold reduction at ages 20–24 might reflect the combined effects of reduced aflatoxin exposure and partial neonatal HBV vaccination. Decrease incidence in age groups >25 years could mainly be attributable to rapid aflatoxin reduction. Compared with 1980–83, liver cancer incidence in 1990–93 significantly decreased 3.4-fold at ages 20–24, and 1.9-fold at ages 25–29 when the first vaccinees were

BACKGROUND Hepatocellular carcinoma (HCC) is generally considered a hypervascular tumor when visualized by angiography. However, small HCCs are not always found to be hypervascular. METHODS To evaluate this, 50 HCCs < or =3 cm in diameter were studied. The 50 tumors consisted of 16 well-differentiated HCCs, 25 moderately differentiated HCCs, and 9 that were each a mixture of well- and moderately differentiated HCC. RESULTS The mean number of portal tracts in the well-differentiated HCCs was 34% of the number in the surrounding nontumorous liver, and few intratumoral arterioles were seen. In contrast, the mean number of portal tracts in the moderately differentiated HCCs was 0.6% of the number in the surrounding nontumorous liver, and abundant intratumoral arterioles were seen. For HCCs that contained both well-differentiated and moderately differentiated tumor, the distribution of portal tracts and intratumoral arterioles in each portion was similar to that seen in well-differentiated or moderately differentiated HCC alone, respectively. HCCs that were larger than 1.5 cm in diameter had fewer portal tracts and more intratumoral arterioles than HCCs whose diameters were < or =1.5 cm. CONCLUSIONS As small HCCs increase in size and become increasingly dedifferentiated, the number of portal tracts apparently decreases and intratumoral arterioles develop. These findings may reflect changes in the hemodynamics as the HCC develops.

Two patients with hepatocellular carcinoma (HCC) were identified who had substantial deletions within the hepatitis B virus (HBV) X gene from HCC tissues. In one patient, the deletion was found at nt. 382-389 (codons 128-130) of the X gene, followed by two nucleotide substitutions, a frame shift, and formation of a new stop codon. In the second patient, the deletion was found at nt. 389-396 (codons 130-132) of the X gene, followed by one nucleotide substitution, a frame shift, and formation of a new stop codon. The resulting X proteins in both cases would be truncated at the 3′ end and would be 20 amino acids shorter than the full length X protein. These patients had been identified during a study of 25 HCC patients from Qidong, China in whom a 228-base region of the X gene was sequenced. No deletions were found within this X gene sequence in HCC tissues from the other 23 patients or in the 20 adjacent noncancerous liver samples available from these patients. However, the fact that these deletions encompassed codons 130 and 131, two adjacent codons where point mutations were found in 21 of the remaining 23 patients, suggests that this region may play an important role in hepatocarcinogenesis.

Expression of transforming growth factor-alpha (TGF-alpha) and its receptor, the epidermal growth factor receptor (EGFR), in hepatocellular carcinomas (HCCs) and adjacent nontumorous livers from 25 Japanese patients were examined using immunoperoxidase staining of paraffin-embedded sections. TGF-alpha was detected in 24 of 25 (96%) HCCs and 23 of 24 (96%) available adjacent nontumorous livers. EGFR was detected in 16 of 25 (64%) HCCs and 17 of 24 (71%) adjacent nontumorous livers. TGF-alpha and EGFR were not detected by immunohistochemical staining in normal livers. Fifteen of 25 HCCs contained an apparent area of a second tumor (two of the 15 also contained a third tumor) that had a less-differentiated histological grade developing within or adjacent to the first tumor. In those cases, staining in the less-differentiated area of tumor was usually less intense than in the more highly differentiated area (80% of cases for TGF-alpha; 91% for EGFR). These data confirm that increased expression of TGF-alpha and EGFR occur frequently in human HCC. Furthermore, the detection of greater staining in more highly differentiated portions of the tumors suggests that increased expression of TGF-alpha and EGFR may be events of the early stages of human hepatocarcinogenesis.

Recent studies have identified epithelial cell populations in human livers that are similar to the "oval cells" and "transitional cells" seen in rat livers during the early stages of chemical carcinogenesis. It has been suggested that these cells might be precursors of hepatocytes and theoretically could be involved in hepatocarcinogenesis. The hepatitis B virus (HBV) also is believed to play a role in the etiology of hepatocellular carcinoma (HCC). Therefore, a study was conducted in nontumorous livers adjacent to HCCs obtained from 26 patients from China to determine whether HBV antigens could be identified in oval cells and transitional cells using an immunohistochemical technique. Hepatitis B surface antigen (HBsAg) was detected in the nontumorous livers of 22/26 (85%) patients. HBsAg was detected in oval cells in 18/26 (69%), in transitional cells in 21/26 (81%), and in mature hepatocytes in 22/26 (85%), but not in bile duct or ductule cells. Transforming growth factor-alpha (TGF-alpha) was expressed in oval cells, transitional cells, and bile duct cells in 24/26 (92%) patients, an in mature hepatocytes in 25/26 (96%). Coexpression of HBsAg and TGF-alpha was identified in the same cells in populations of oval cells and transitional cells of selected patients. Because of the possibility that oval cells could be a source of evolving HCC, these findings suggest that expression of TGF-alpha associated with HBV infection of oval cells could be a mechanism of human hepatocarcinogenesis. Thus, oval cells could be a site (or one of the sites) where HBV participates in the development of HCC.

Background Mutations of the p53 tumor suppressor gene have been reported in 50% of patients with hepatocellular carcinoma (HCC) from China and South Africa. These reports suggested an association of p53 mutations with high levels of aflatoxin in the diet. Most studies of p53 and HCC, however, have not fully evaluated the possible role of the hepatitis B virus (HBV). Aflatoxin is a substance produced by food mold that is known to cause HCC in experimental animals. Purpose The purpose of this study was to evaluate the relationship of p53 gene mutation to high or low levels of aflatoxin in the diet and to HBV infection. Methods p53 protein and hepatitis B surface antigen (HBsAg) were evaluated by immunohistochemistry using the avidin-biotin-peroxidase system in paraffin-embedded specimens of HCC and of adjacent nontumorous liver tissue from 43 patients. Tissue specimens from three normal human livers were also evaluated. HCCs and adjacent nontumorous liver tissues were obtained from 23 patients from Qidong, China, where aflatoxin levels in the diet are high, and from 20 patients from two regions in the United States (patients from the National Institutes of Health, Bethesda, Md., and Kuakini Medical Center, Honolulu, Hawaii), where aflatoxin levels in the diet are low. Results Mutant p53 protein was detected in the nuclei of HCCs from 14 (61%) of 23 patients from China and from three (30%) of 10 patients and six (60%) of 10 patients, respectively, from the two regions of the United States. A statistically significant association between detection of mutant p53 protein in HCC cells and the detection of HBsAg in hepatocytes of the adjacent nontumorous liver tissue was observed in patients from China and the United States considered together. Conclusion Mutations of the tumor suppressor gene p53 in hepatocellular carcinomas are not limited to patients from geographic regions where the ingestion of aflatoxin is high. In many patients, these mutations may be associated with HBV infection. Implications The possible interaction of chronic HBV infection and p53 gene mutation, suggested by these data, indicates a mechanism by which HBV infection beginning early in life could contribute to the subsequent development of HCC.

Studies have suggested that a truncated form of the hepatitis C virus (HCV) core protein can enter hepatocyte nuclei and might play a role in HCV-associated hepato-carcinogenesis. In the present study, the HCV core gene from hepatocellular carcinomas (HCC) and/or adjacent non-tumorous liver tissues from eight patients was amplified by nested RT-PCR and sequenced. Mutations in the HCV core gene that would encode a truncated core protein were found in 4 of the 8 patients. Since truncated core proteins have been shown to be capable of entry into the hepatocyte nucleus (unlike HCV itself, which is an exclusively cytoplasmic virus), the detection of mutated sequences encoding them in these four HCC patients suggests that these mutations may have played a role in the development of these HCCs.

This is the first report that a Fusarium toxin nivalenol (NIV) naturally existing at high levels in dietary food in high-risk areas of cancer of esophagus and gastric cardia in China induced benign and malignant tumors in mice. The levels of two Fusarium toxins, nivalenol and deoxynivalenol (DON) were quantitated using high performance liquid chromatography (HPLC) in a total of 97 samples of dietary wheat flour, barley and corn collected from families in two areas with high mortality rate of cancer of esophagus and gastric cardia (132/100,000), Linxian, Henan province and Cixiang, Hepei province, China. The mean level of NIV and DON in three dietary foods was 830+/-927 microg/kg (range 584-1,780 microg/kg) and 4,281+/-6,114 microg/kg (range 732-10,980 microg/kg) respectively. The highest mean level of NIV was 1,780+/-1,705 microg/kg found in barley from Linxian, that of DON was 10,980+/-10,139 microg/kg found in corn from Cixiang. NIV was undetectable in 2 samples of rice from USA. The mean levels of NIV in three main dietary foods in those two high-risk areas were estimated at 400 to 800-fold higher than that in the USA, where NIV was undetectable in dietary food, and the mortality rate of esophageal cancer is

There is evidence to suggest that a focus of less-differentiated hepatocellular carcinoma (HCC) may arise within a pre-existing well-differentiated HCC, eventually replacing it. In the present study, the p53 tumor suppressor gene was analyzed by immunohistochemistry in 31 hepato-cellular carcinomas (HCCs) containing two or more regions in the same nodule with different histologic grades. p53 was overexpressed in the nucleus in 13 of 31 HCCs (42%), in seven of which p53 overexpression was seen only in the less-differentiated area of the tumor. This suggests that overexpression of presumed mutant p53 may have contributed to dedifferentiation during the development of HCC.

p53 expression was studied by immunohistochemical methods in benign and malignant human epithelial liver lesions in 46 patients from Hungary. Positive immunostaining for p53 protein, indicating the overexpression or prolonged half-life of p53 protein, was detected in the nuclei of tumour cells of seven of the 16 hepatocellular carcinomas (HCC) (44%), including three HCC patients with hepatitis B virus infection. Immunostaining of p53 was seen in one of the seven hepatoblastomas, none of the 17 focal nodular hyperplasias, and none of the six hepatocellular adenomas. The detection of p53 in a relatively high percentage of the HCC cases in Hungary, a country in which aflatoxin contamination of the diet is rare, suggests that factors other than aflatoxin led to the accumulation or overexpression of p53 in these patients.

The expression of transforming growth factor-alpha (TGF-alpha) was studied in 33 surgically excised human hepatocellular carcinomas (HCCs), focal nodular hyperplasias (FNHs), and the surrounding liver tissue from patients who were life-long residents of Hungary. Monoclonal antibodies were used to localize TGF-alpha and hepatitis B surface antigen (HBsAg) using an avidin-biotin-peroxidase immunohistochemical method on paraffin-embedded sections. Transforming growth factor-alpha was detected in the tumor tissue in 13 of 16 patients with HCC (81%) and in 12 of 17 patients with FNH (71%). Three patients with HCC were actively infected with hepatitis B virus, indicated by the detection of HBsAg in the serum and in adjacent nontumorous liver. Transforming growth factor-alpha was detected in the same nontumorous hepatocytes as HBsAg, often in areas of the hepatocyte cytoplasm, with a "ground glass" appearance. Bile duct cells were stained for TGF-alpha in the surrounding nontumorous liver tissue and a more intensive immunostaining was observed in the proliferating bile ducts in the FNH cases, which suggests that TGF-alpha may participate in the growth regulation of bile ducts.

A well-differentiated trabecular hepatocellular carcinoma (HCC) and a well-differentiated tumor resembling HCC from each of two chimpanzees were found to have histochemical and immunohistochemical staining characteristics similar to those in human HCCs. Transforming growth factor alpha was overexpressed in both tumors. Oval cells, thought to be liver stem cell progeny with a possible role in hepatocarcinogenesis, were observed among nontumorous hepatocytes, particularly near the tumors. Hepatic tumors are rare in chimpanzees but their similarities to human HCC provides a useful research model.

Proliferation of a new population of epithelial cells with distinct structure, as well as cytokeratin and alpha-fetoprotein expression, was observed in nonneoplastic liver tissues from 14 cases (13 hepatitis B virus-positive) of human hepatocellular carcinoma. These cells were characterized by oval nuclei; scant, pale cytoplasm; small cell size; and cross-reaction with a monoclonal antibody against rat oval cells. These putative human oval cells were strongly positive for cytokeratin 19 and displayed considerable heterogeneity in alpha-fetoprotein and albumin expression. The oval cells were most prominent in actively regenerating nodules and in liver tissue surrounding the cancer. Oval cells and transitional types of cells appear to be the principal producers of alpha-fetoprotein in the regenerating liver. Cancer cells positive for cytokeratins 8, 18 and 19 were observed in half the hepatocellular carcinomas studied. The data suggest that a new cell population structurally similar to oval cells seen in early stages of chemical hepatocarcinogenesis in rats is consistently present in regenerating liver lesions associated with human hepatocellular carcinoma. Furthermore, it is possible that the proliferation of these oval-type cells may partly account for the elevation of serum alpha-fetoprotein frequently seen in precancerous stages of hepatitis B virus-associated human hepatocellular carcinoma.

The cellular distribution and temporal expression of transcripts from transforming growth factor-beta 1 (TGF-beta 1) and procollagen alpha 1(I), alpha 1(III), and alpha 1(IV) genes were studied in carbon tetrachloride (CCl4)-induced rat liver fibrosis by using in situ hybridization technique. During the fibrotic process, TGF-beta 1 and procollagen genes were similarly and predominantly expressed in Desmin-positive perisinusoidal cells (e.g., fat-storing cells and myofibroblasts) and fibroblasts and their expression continued to be higher than those observed in control rats. These transcripts were also observed in inflammatory cells mainly granulocytes and macrophage-like cells at the early stages of liver fibrosis. The production of extracellular matrix along small blood vessels and fibrous septa coincided with the expression of these genes. Expression of TGF-beta 1 and procollagen genes were not detected in hepatocytes throughout the experiment. No significant differences in cellular distribution or time course of gene expression among procollagen alpha 1(I), alpha 1(III), and alpha 1(IV) were observed. Desmin-positive perisinusoidal cells and fibroblasts appeared to play the principal role in synthesis of collagens in CCl4-induced hepatic fibrosis. The simultaneous expression of TGF-beta 1 and procollagen genes in mesenchymal cells, including Desmin-positive perisinusoidal cells, during hepatic fibrosis suggests the possibility that TGF-beta 1 may have an important role in the production of fibrosis.

Cultured fetal human stomach, esophagus and liver activated benzo[a]pyrene (BP), aflatoxin B1 (AFB) and certain N-nitrosamines into metabolites that bound to cellular DNA. When the 3 organs were compared the highest level of activity was observed in the stomach. The interindividual variation was 10-fold and the amount of carcinogen-DNA adducts did not correlate with the sex or age of the fetus. The reaction products between BP or AFB and cellular DNA were investigated in liver explants. The carcinogen-DNA adduct patterns were identical to those observed in adult human tissues; BPDEI-Gua being the major adduct formed by BP and 2,3-dihydro-2-(7′-guanyl)-3-hydroxy-AFB by AFB. The results indicate that fetal organs can metabolize those oncogenic compounds at an early stage of the development, and that the metabolic pathways and DNA adducts are quite similar to those in experimental animals in which the compounds are carcinogenic.