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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

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminated the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients.

In a 30-year follow-up of the Qidong Hepatitis B Intervention Study, Yawei Zhang and colleagues examine the effects of neonatal vaccination on liver diseases. Please see later in the article for the Editors' Summary, Background Neonatal hepatitis B vaccination has been implemented worldwide to prevent hepatitis B virus (HBV) infections. Its long-term protective efficacy on primary liver cancer (PLC) and other liver diseases has not been fully examined. Methods and Findings The Qidong Hepatitis B Intervention Study, a population-based, cluster randomized, controlled trial between 1985 and 1990 in Qidong, China, included 39,292 newborns who were randomly assigned to the vaccination group in which 38,366 participants completed the HBV vaccination series and 34,441 newborns who were randomly assigned to the control group in which the participants received neither a vaccine nor a placebo. However, 23,368 (67.8%) participants in the control group received catch-up vaccination at age 10–14 years. By December 2013, a total of 3,895 (10.2%) in the vaccination group and 3,898 (11.3%) in the control group were lost to follow-up. Information on PLC incidence and liver disease mortality were collected through linkage of all remaining cohort members to a well-established population-based tumor registry until December 31, 2013. Two cross-sectional surveys on HBV surface antigen (HBsAg) seroprevalence were conducted in 1996–2000 and 2008–2012. The participation rates of the two surveys were 57.5% (21,770) and 50.7% (17,204) in the vaccination group and 36.3% (12,184) and 58.6% (17,395) in the control group, respectively. Using intention-to-treat analysis, we found that the incidence rate of PLC and the mortality rates of severe end-stage liver diseases and infant fulminant hepatitis were significantly lower in the vaccination group than the control group with efficacies of 84% (95% CI 23%–97%), 70% (95% CI 15%–89%), and 69% (95% CI 34%–85%), respectively. The estimated efficacy of catch-up vaccination on HBsAg seroprevalence in early adulthood was 21% (95% CI 10%–30%), substantially weaker than that of the neonatal vaccination (72%, 95% CI 68%–75%). Receiving a booster at age 10–14 years decreased HBsAg seroprevalence if participants were born to HBsAg-positive mothers (hazard ratio [HR] = 0.68, 95% CI 0.47–0.97). Limitations to consider in interpreting the study results include the small number of individuals with PLC, participants lost to follow-up, and the large proportion of participants who did not provide serum samples at follow-up. Conclusions Neonatal HBV vaccination was found to significantly decrease HBsAg seroprevalence in childhood through young adulthood and subsequently reduce the risk of PLC and other liver diseases in young adults in rural China. The findings underscore the importance of neonatal HBV vaccination. Our results also suggest that an adolescence booster should be considered in individuals born to HBsAg-positive mothers and who have completed the HBV neonatal vaccination series. Please see later in the article for the Editors' Summary, Editors' Summary Background Hepatitis B is a life-threatening liver infection caused by the hepatitis B virus (HBV). HBV, which is transmitted through contact with the blood or other bodily fluids of an infected person, can cause both acute (short-term) and chronic (long-term) liver infections. Acute infections rarely cause any symptoms and more than 90% of adults who become infected with HBV (usually through sexual intercourse with an infected partner or through the use of contaminated needles) are virus-free within 6 months. However, in sub-Saharan Africa, East Asia, and other regions where HBV infection is common, HBV is usually transmitted from mother to child at birth or between individuals during early childhood and, unfortunately, most infants who are infected with HBV during the first year of life and many children who are infected before the age of 6 years develop a chronic HBV infection. Such infections can cause liver cancer, liver cirrhosis (scarring of the liver), and other fatal liver diseases. In addition, HBV infection around the time of birth can cause infant fulminant hepatitis, a rare but frequently fatal condition. Why Was This Study Done? HBV infections kill about 780,000 people worldwide annually but can be prevented by neonatal vaccination—immunization against HBV at birth. A vaccine against HBV became available in 1982 and many countries now include HBV vaccination at birth followed by additional vaccine doses during early childhood in their national vaccination programs. But, although HBV vaccination has greatly reduced the rate of chronic HBV infection, the protective efficacy of neonatal HBV vaccination against liver diseases has not been fully examined. Here, the researchers investigate how well neonatal HBV vaccination protects against primary liver cancer and other liver diseases by undertaking a 30-year follow-up of the Qidong Hepatitis B intervention Study (QHBIS). This cluster randomized controlled trial of neonatal HBV vaccination was conducted between 1983 and 1990 in Qidong County, a rural area in China with a high incidence of HBV-related primary liver cancer and other liver diseases. A cluster randomized controlled trial compares outcomes in groups of people (towns in this study) chosen at random to receive an intervention or a control treatment (here, vaccination or no vaccination; this study design was ethically acceptable during the 1980s when HBV vaccination was unavailable in rural China but would be unethical nowadays). What Did the Researchers Do and Find? The QHBIS assigned nearly 80,000 newborns to receive either a full course of HBV vaccinations (the vaccination group) or no vaccination (the control group); two-thirds of the control group participants received a catch-up vaccination at age 10–14 years. The researchers obtained data on how many trial participants developed primary liver cancer or died from a liver disease during the follow-up period from a population-based tumor registry. They also obtained information on HBsAg seroprevalence—the presence of HBsAg (an HBV surface protein) in the blood of the participants, an indicator of current HBV infection—from surveys undertaken in1996–2000 and 2008–2012. The researchers estimate that the protective efficacy of vaccination was 84% for primary liver cancer (vaccination reduced the incidence of liver cancer by 84%), 70% for death from liver diseases, and 69% for the incidence of infant fulminant hepatitis. Overall, the efficacy of catch-up vaccination on HBsAg seroprevalence in early adulthood was weak compared with neonatal vaccination (21% versus 72%). Notably, receiving a booster vaccination at age 10–14 years decreased HBsAg seroprevalence among participants who were born to HBsAg-positive mothers. What Do These Findings Mean? The small number of cases of primary liver cancer and other liver diseases observed during the 30-year follow-up, the length of follow-up, and the availability of incomplete data on seroprevalence all limit the accuracy of these findings. Nevertheless, these findings indicate that neonatal HBV vaccination greatly reduced HBsAg seroprevalence (an indicator of current HBV infection) in childhood and young adulthood and subsequently reduced the risk of liver cancer and other liver diseases in young adults. These findings therefore support the importance of neonatal HBV vaccination. In addition, they suggest that booster vaccination during adolescence might consolidate the efficacy of neonatal vaccination among individuals who were born to HBsAg-positive mothers, a suggestion that needs to be confirmed in randomized controlled trials before booster vaccines are introduced into vaccination programs. Additional Information Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001774. The World Health Organization provides a fact sheet about hepatitis B (available in several languages) and information about hepatitis B vaccination The World Hepatitis Alliance (an international not-for-profit, non-governmental organization) provides information about viral hepatitis, including some personal stories about hepatitis B from Bangladesh, Pakistan, the Philippines, and Malawi The UK National Health Service Choices website provides information about hepatitis B The not-for-profit British Liver Trust provides information about hepatitis B, including Hepatitis B: PATH B, an interactive educational resource designed to improve the lives of people living with chronic hepatitis B MedlinePlus provides links to other resources about hepatitis B (in English and Spanish) Information about the Qidong Hepatitis B intervention Study is available Chinese Center for Disease Control and Prevention provides links about hepatitis B prevention in Chinese

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.

In order to study the mechanisms by which p53 function is regulated, human wild-type p53 cDNA was cloned into a vaccinia virus vector and the expressed p53 protein was used to investigate binding of the p53 by cellular proteins from a cDNA expression library from human liver. One protein that bound wild-type p53 had >99% homology with DNA topoisomerase IIb. p53 protein was coimmunoprecipitated from topoisomerase II-rich cell lysates (but not from topoisomerase II-deficient cell lysates) by an antibody to topoisomerase IIa and IIb. This binding was shown to occur without a dsDNA intermediary. Hepatocellular carcinomas (HCCs) and adjacent nontumorous liver tissues from ten patients were studied to determine the level of expression of topoisomerase II and p53. Overexpressed topoisomerase II proteins were detected by western blot in six of ten HCCs (60%), including several in which presumed wild-type p53 was detected by immunohistochemistry. No topoisomerase II expression was detectable in the ten nontumorous liver tissues from the same patients or in a sample of normal human liver.

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.

The effect of interferon alfa (IFN-α) therapy on the expression of transforming growth factor alpha (TGF-α) in the liver during chronic hepatitis B was investigated. Serial liver biopsy specimens were evaluated from 35 patients who had participated in a randomized, controlled trial of recombinant human IFN-α for the treatment of chronic hepatitis B. Percutaneous liver biopsy specimens obtained before and 1 year after entry in the trial were sectioned and stained with a monoclonal antibody to TGF-α in an avidin-biotin-peroxidase-complex system. The expression of TGF-α in each section was evaluated blindly (with respect to treatment group and order of biopsies) and was numerically scored. There was no significant difference in TGF-α expression before or after therapy between 13 patients receiving daily IFN-α, 13 receiving alternate-day IFN-α, and 9 receiving no therapy. Sustained clearance of HBV-DNA and DNA polymerase activity occurred in 8 of 26 treated patients (“responders”); the 18 other patients were “nonresponders”. Expression of TGF-α before IFN-α therapy was significantly higher in responders than in nonresponders; after IFN-α therapy, TGF-α expression decreased significantly among responders compared with nonresponders and untreated controls. Thus, the level of expression of TGF-α in the liver, which was correlated with the severity of inflammation in the liver in this study, appeared to be predictive of the response to IFN-α therapy in chronic hepatitis B, with a higher level of expression indicating a greater likelihood that the patient would respond.

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.

BACKGROUND Hepatitis B virus (HBV) infection is closely associated with the development of hepatocellular carcinoma (HCC) in many patients, but the mechanisms by which HBV contributes to HCC are not known. Transforming growth factor-alpha (TGF-alpha), a regulator of growth and regeneration in rat liver that can be found in high levels in some human cancers, theoretically could play such an intermediate role in the development of HCC. METHODS The expression of TGF-alpha and its relation to the HBV antigens were evaluated in human HCC and adjacent nontumorous livers from 33 patients from the United States and China using immunoperoxidase staining of paraffin-embedded sections. RESULTS TGF-alpha was detected in HCC from 27 of 33 (82%) patients; the frequencies were similar in patients from the United States and China. TGF-alpha was detected in HCC more frequently in patients whose adjacent nontumorous livers had detectable hepatitis B surface antigen (HBsAg) and/or hepatitis B core antigen (HBcAg) than in those whose adjacent livers lacked HBsAg and HBcAg. Detection of TGF-alpha was not affected by tumor size, histologic type, or grade. TGF-alpha was detected in adjacent nontumorous livers from 31 of 33 patients (94%). Coexpression at a high intensity of TGF-alpha and HBsAg in the same hepatocytes could be demonstrated by specific staining of consecutively cut sections for 17 of 33 patients (52%). CONCLUSIONS TGF-alpha is expressed at a high level in 82% of human HCC. Localization of HBsAg within the same hepatocytes as TGF-alpha suggests a possible interaction between HBV and TGF-alpha during hepatocarcinogenesis in humans. Stimulation of TGF-alpha expression could be part of a chain of events by which HBV contributes to the development of HCC in some patients.

Hepatitis B virus (HBV) infections are still a major health issue, with approximately 350 million people chronically infected with HBV worldwide. Information about the minimum copy number of HBV genomes required for infection would be useful as a reference for drug and vaccine development; for monitoring HBV patients during treatment; for screening of blood, organ, and tissue donors; and for regulating nucleic acid amplification assays for HBV.Serum samples from chronic carriers (hepatitis B surface antigen-positive and antibody to HBV core antigen-positive) of the three most common subtypes of HBV were studied; their infectivity titers had been evaluated previously in chimpanzees. The genotypes of the HBV samples were determined by DNA sequences and type-specific amino acids of the S gene of HBV. Copy numbers of HBV DNA were quantified by real-time TaqMan polymerase chain reaction (PCR) and by nested PCR applied to limiting dilutions. The copy number determined for each inoculum was compared with previously defined chimpanzee infectivity titers.The genotypes of the HBV adw, ayw, and adr inocula were A, D, and C, respectively. The concentration of HBV DNA was determined to be 5.4 x 10(9), 2.5 x 10(9), and 3.1 x 10(8) genome equivalents (geq) per mL for serum samples containing the adw, ayw, and adr, respectively. The chimpanzee infectivity titers per milliliter of these initial HBV-containing serum samples were previously determined to be 10(7.5) for adw, 10(7.5) for ayw (MS-2 strain), and 10(8) for adr.The minimal copy number of HBV DNA in chronic carriers of HBV that can infect the chimpanzee model was estimated to be from 3 to 169 geq based upon the three well-characterized inocula.

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

BACKGROUND: A study was designed to estimate relative analytic sensitivity and window-period (WP) closure and to project incremental yield of newer HBsAg tests, pooled-sample NAT, and single-sample NAT, compared to currently licensed HBsAg tests. STUDY DESIGN AND METHODS: HBV DNA and HBsAg test results for 23 HBV seroconversion (SC) panels were first analyzed to construct a model of primary HBV viremia. One-hundred representative samples were then selected from 10 panels and coded with 28 analytical controls. All 128 samples were tested by seven HBsAg tests and by four pooled-sample and three single-sample NAT assay formats. Results were analyzed to obtain differential times to HBV detection and combined with HBV incidence rates to project comparative yields. RESULTS: HBV doubling time during the ramp-up phase was estimated at 2.56 days. HBsAg concentrations at cutoff for new tests ranged from 0.07 to 0.12 ng per mL, compared with 0.13 to 0.62 ng per mL for licensed tests. Estimated viral load at cutoff ranged from 102 to 267 IU per mL for new tests and from 363 to 1069 IU per mL for licensed tests. HBsAg tests detected 31 to 63 percent of early ramp-up phase samples in the 100-member seroconversion panel study, while pooled-sample NAT detected 55 to 71 percent and single-sample NAT, 82 to 99 percent. Compared with currently licensed HBsAg assays, newer HBsAg assays would reduce the WP by 2 to 9 days; pooled-sample NAT would reduce the WP by 9 to 11 days; and single-sample NAT would reduce the WP by 25 to 36 days. CONCLUSION: Newer HBsAg tests would be expected to detect an additional 15 to 21 infected units per 107 donations, compared to licensed HBsAg tests. Sensitivity, WP closure, and yield projections for newer HBsAg assays and pooled-sample NAT are comparable. Single-sample NAT would increase yield by 13 to 15 units per 107 donations over pooled-sample NAT and newer HBsAg assays and by 35 to 50 units per 107 donations over currently licensed HBsAg assays.

The expression of transforming growth factor alpha (TGF-alpha) was examined in a human hepatoblastoma cell line, Hep G2, which does not contain hepatitis B virus (HBV) DNA, and in the cell line 2.2.15, which was formed by the transfection of Hep G2 cells with the complete HBV DNA, to study the possibility that HBV and TGF-alpha could function as co-factors in hepatocarcinogenesis. Northern blot hybridization of RNA extracted from these cell lines, with densitometric analysis, revealed expression of the TGF-alpha gene in the transfected cells at a level three times higher than in the nontransfected cells. Staining of the cells using a monoclonal antibody to TGF-alpha and the avidin-biotin-peroxidase immunohistochemical method revealed a much higher intensity of TGF-alpha staining in the transfected cell line. These findings show that the presence of HBV DNA appears to cause a significant up-regulation of the TGF-alpha gene. This effect on the TGF-alpha gene may be a mechanism by which HBV contributes to the etiology of hepatocellular carcinoma in some patients.

Mutations of the p53 tumor suppressor gene are common in hepatocellular carcinomas (HCCs). Detection of mutations by sequencing provides more information than immunohistochemical staining, but the equipment needed and the time required make it less practical for use in large-scale studies or in studies in developing countries. The degree of correlation between results obtained with these two methods has been studied in various tumors but has not been well-established in human HCCs. Paraffin sections of HCCs of 28 patients from Qidong, China were immunohistochemically stained using monoclonal antibody to p53. In addition, exons 5-8 of the p53 gene were sequenced in these HCCs. Of the 28 HCCs, nine had 0-9% of nuclei stained for p53, and 19 had 50-95% stained. Mutations in p53 exons 5-8 were found in 17/28 (61%) HCCs, including 15 at codon 249 (exon 7), one at codon 198 (exon 6), and one at codon 175 (exon 5). Among these 17 cases with p53 mutations, 16 cases (94%) had 50-95% of nuclei stained. Among 11 HCCs with no mutations by sequencing, 8 were also negative by immunohistochemistry (0-9% of nuclei stained) (73%) (the five HCCs with no staining whatsoever all had wild-type p53). Immunohistochemical staining to detect p53 mutations in human HCCs detected most mutations that were detected by sequencing (94% sensitivity, 73% specificity), and this method is therefore suitable when sequencing cannot be performed.

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.

Alterations in the expression of the RB tumour suppressor gene were found by Western immunoblot in three of seven hepatocellular carcinoma and hepatoblastoma cell lines. Abnormalities were detected by single-strand conformation polymorphism (SSCP) within exons 17-21 in RB cDNA from two of these three cell lines and within exons 20-21 in the third cell line. In these three cell lines with abnormal RB expression, abnormal expression of the p53 tumour suppressor gene was also found, apparently the product of a mutant gene. Thus, mutations within the RB gene (or splice-site mutations with exon-skipping) and apparent mutations of the p53 gene together may have contributed to the development of three of these tumours or to the establishment of these cell lines.

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.

In an area with high incidence of esophageal cancer, Linxian, staple food is heavily contaminated by fungi of the Fusarium genus. We have found that T-2 toxin produced by Fusarium has both direct cytotoxic and proliferative effects on fetal esophageal epithelium. At higher dosage of T-2 toxin (4 ng/ml for 6 days) the cultured epithelium became necrotic. At a lower dosage range of 0.2-1.2 ng/ml, T-2 toxin caused mitogenic effects including focal basal cell hyperplasia, dysplasia, and increased number of mitoses. Atypical mitoses were also seen. These changes are very similar to the premalignant lesions seen in epithelium adjacent to human esophageal carcinoma. These observations suggest that Fusarium mycotoxin can have a role in human esophageal carcinogenesis and should be further investigated.

T-2 toxin is an important representative of trichothecenes produced by various species of imperfect fungi, mainly Fusarium genus. It is a naturally occurring toxin that can cause severe diseases in human and animals. No definite data demonstrating the carcinogenic potential of T-2 toxin had been reported up to now. We demonstrated that T-2 toxin reproducibly induced chromosomal structural aberrations both in cultured human peripheral blood lymphocytes as well as in V79 Chinese hamster fibroblasts. The mean percentage of cells with aberration of human lymphocytes from normal individuals induced by T-2 toxin is 49-fold (9.8%) of the mean percentage of corresponding control cultures without T-2 toxin (0.2%). T-2 toxin induced chromosome type (76%) as well as chromatid type (24%) of aberrations; among them, acentric fragment (46%) was the most common type, and chromatid gap, deletion, and chromosome gap were the next most common. T-2 toxin can induce aberrations in cells at different phases of the cell cycle. There are definite dose-effect relationships within a certain range of dosage of T-2 toxin in experiments with both human peripheral blood lymphocytes and V79 cells. T-2 toxin exhibited three types of effects on cells, namely, mitogenic at lowest concentration, clastogenic (chromosome aberration) at median concentration, and cytotoxic at higher concentration. The dose-effect curves of these three effects are partly overlapping. There is some interindividual difference in sensitivity of response for the clastogenic effect of T-2 toxin, but no resistant individual was observed. Sex or age effect was not observed. The above results suggest that T-2 toxin has carcinogenic potentials. The dosage of aflatoxin that can induce chromosomal aberration of human peripheral blood lymphocytes is thousands-fold of the dosage of T-2 toxin as shown in this report. T-2 toxin might have more hazardous potentials than had been previously considered. The carcinogenic potential of T-2 toxin and related trichothecenes and their possible roles in carcinogenesis of upper gastrointestinal (G.I.) tract cancers need to be further investigated.