Preventing primary liver cancer: the HBV vaccination project in the Gambia (West Africa).

The Gambia Hepatitis Intervention Study (GHIS) consisted in the progressive introduction of HBV plasma-derived vaccine in different zones of this African country during the period 1986-1990. The study was launched and coordinated by IARC and is one of the most effective examples of an intervention project that both substantially contributed to our knowledge and to the health of local populations. Similar intervention studies have been carried out in South-East Asia. The studies indicate that the natural history of HBV infection differs in different populations , having a direct relevance for the implementation of HBV vaccination programmes in various parts of the world.

20 years into the Gambia Hepatitis Intervention Study: assessment of initial hypotheses and prospects for evaluation of protective effectiveness against liver cancer.

Primary hepatocellular carcinoma is the commonest cancer in The Gambia. The Gambia Hepatitis Intervention Study (GHIS) was established in 1986 to evaluate the protective effectiveness of infant hepatitis B immunization in the prevention of chronic liver disease, particularly, hepatocellular carcinoma and cirrhosis later in adult life. This program was designed based on a series of assumptions. Here, we used data from observational and epidemiologic studies developed since 1986 to examine the validity of these assumptions. We found that (a) hepatitis B vaccine coverage was 15% more than originally assumed, (b) protection against hepatitis B virus (HBV) infection was not dependent on the number of vaccine doses received, (c) perinatal infection with HBV was of negligible importance, and (d) the HBV attributable risk of hepatocellular carcinoma at age < 50 was 70% to 80%, lower than initially assumed. Based on these data, the final outcome of the GHIS should be measurable from 2017, sooner than originally assumed. The GHIS strategy takes into account-specific patterns of virus epidemiology and natural history of hepatocellular carcinoma in Africa and provides a model for integrating and evaluating new vaccines into the Expanded Programme of Immunization of sub-Saharan African countries.

249(ser) TP53 mutation in plasma DNA, hepatitis B viral infection, and risk of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) from regions with high dietary exposure to aflatoxins and endemic for hepatitis B virus (HBV) often contain a specific mutation at codon 249 in TP53 (249(ser); AGG to AGT, Arg to Ser). This mutation is also detectable in circulating cell-free DNA from the plasma of HCC patients and healthy subjects in these regions. We have examined the joint effect of plasma 249(ser) and HBV infection in a case-control study design involving 348 control, 98 cirrhotic, and 186 HCC participants from The Gambia, West Africa, an area of high HCC incidence. The 249(ser) mutation was detected in 3.5% of controls, 15.3% of cirrhotics, and 39.8% of HCC cases (adjusted odds ratios (OR): 4.83, (95% confidence interval (CI): 1.71-13.7) for cirrhosis and 20.3 (8.19-50.0) for HCC). HBsAg positivity along with plasma 249(ser) was observed in 45/183 (24.6%) HCC cases compared to only one (0.3%) control. Risk for HCC was associated with markers of HBV alone (OR: 10.0, 95% CI: 5.16-19.6), 249(ser) alone (OR: 13.2, 95% CI: 4.99-35.0), and both markers present (OR: 399, 95% CI: 48.6-3270). These results suggest a multiplicative effect on HCC risk resulting from the mutational effect of aflatoxin on TP53, as monitored by detection of plasma 249(ser), with concomitant chronic infection with HBV.

Quantitative analysis of plasma TP53 249Ser-mutated DNA by electrospray ionization mass spectrometry.

A mutation in codon 249 of the TP53 gene (249(Ser)), related to aflatoxin B(1) exposure, has previously been associated with hepatocellular carcinoma risk. Using a novel internal standard plasmid, plasma concentrations of 249(Ser)-mutated DNA were quantified by electrospray ionization mass spectrometry in 89 hepatocellular carcinoma cases, 42 cirrhotic patients, and 131 nonliver diseased control subjects, all from highly aflatoxin-exposed regions of The Gambia. The hepatocellular carcinoma cases had higher median plasma concentrations of 249(Ser) (2,800 copies/mL; interquartile range: 500-11,000) compared with either cirrhotic (500 copies/mL; interquartile range: 500-2,600) or control subjects (500 copies/mL; interquartile range: 500-2,000; P < 0.05). About half (52%) of the hepatocellular carcinoma cases had >2,500 copies of 249(Ser)/mL plasma, corresponding to the prevalence of this mutation in liver tumors in The Gambia. In comparison, only 15% of control group and 26% of cirrhotic participants exceeded this level (P < 0.05). Further subset analysis revealed a statistically significant, quantitative relation between diagnosis of hepatocellular carcinoma and levels of 249(Ser) detected at 2,501 to 10,000 copies/mL plasma (odds ratio, 3.8; 95% confidence interval, 1.3-10.9) and at >10,000 copies/mL plasma (odds ratio, 62; 95% confidence interval, 4.7-820) when compared with control subjects and after adjusting for age, gender, recruitment site, hepatitis B and C serologic status, and total DNA concentration. Levels of >10,000 copies of 249(Ser)/mL plasma were also significantly associated with the diagnosis of hepatocellular carcinoma (odds ratio, 15; 95% confidence interval, 1.6-140) when compared with cirrhotic patients. Potential applications for the quantification of 249(Ser) DNA in plasma include estimation of long-term, cumulative aflatoxin exposure and selection of appropriate high-risk individuals for targeted intervention.

Hepatocellular carcinoma and polymorphisms in carcinogen-metabolizing and DNA repair enzymes in a population with aflatoxin exposure and hepatitis B virus endemicity.

High rates of hepatocellular carcinoma (HCC) in The Gambia, West Africa, are primarily due to a high prevalence of chronic hepatitis B virus infection and heavy aflatoxin exposure via groundnut consumption. We investigated genetic polymorphisms in carcinogen-metabolizing (GSTM1, GSTT1, HYL1*2) and DNA repair (XRCC1) enzymes in a hospital-based case-control study. Incident HCC cases (n = 216) were compared with frequency-matched controls (n = 408) with no clinically apparent liver disease. Although the prevalence of variant genotypes was generally low, in multivariable analysis (adjusting for demographic factors, hepatitis B virus, hepatitis C virus, and TP53 status), the GSTM1-null genotype [odds ratio (OR), 2.45; 95% confidence interval (95% CI), 1.21-4.95] and the heterozygote XRCC1-399 AG genotype (OR, 3.18; 95% CI, 1.35-7.51) were significantly associated with HCC. A weak association of the HYL1*2 polymorphism with HCC was observed but did not reach statistical significance. GSTT1 was not associated with HCC. The risk for HCC with null GSTM1 was most prominent among those with the highest groundnut consumption (OR, 4.67; 95% CI, 1.45-15.1) and was not evident among those with less than the mean groundnut intake (OR, 0.64; 95% CI, 0.20-2.02). Among participants who had all three suspected aflatoxin-related high-risk genotypes [GSTM1 null, HLY1*2 (HY/HH), and XRCC1 (AG/GG)], a significant 15-fold increased risk of HCC was observed albeit with imprecise estimates (OR, 14.7; 95% CI, 1.27-169). Our findings suggest that genetic modulation of carcinogen metabolism and DNA repair can alter susceptibility to HCC and that these effects may be modified by environmental factors.

A model of interaction: aflatoxins and hepatitis viruses in liver cancer aetiology and prevention.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and has an extremely poor prognosis. The majority of cases occur in south-east Asia and sub-Saharan Africa where the major risk factors are chronic infection with hepatitis B and C viruses (HBV and HCV) as well as dietary exposure to aflatoxins. Aflatoxin B1, the most commonly occurring and potent of the aflatoxins is associated with a specific AGG to AGT transversion mutation at codon 249 of the p53 gene in human HCC, providing mechanistic support to a causal link between exposure and disease. Prospective epidemiological studies have shown a more than multiplicative interaction between HBV and aflatoxins in terms of HCC risk. However, the biology underlying this statistical interaction is not fully understood. There are a number of potential mechanisms including, among others: the fixation of AFB1-induced mutations in the presence of liver regeneration and hyperplasia induced by chronic HBV infection; the predisposition of HBV-infected hepatocytes to aflatoxin-induced DNA damage; an increase in susceptibility to chronic HBV infection in aflatoxin-exposed individuals; and oxidative stress exacerbated by co-exposure to aflatoxins and chronic hepatitis infection. Priorities for prevention are global HBV vaccination, primary and secondary prevention strategies against aflatoxin and the avoidance of transmission of HCV through good hygiene practices.

Molecular epidemiology of human liver cancer: insights into etiology, pathogenesis and prevention from The Gambia, West Africa.

Human liver cancer, primarily hepatocellular carcinoma (HCC), is both common and lethal. Notable variation in HCC incidence rates worldwide corresponds to the prevalence and pattern of the primary etiologic factors. In summary of decades of collaborative research centered in The Gambia, West Africa, this review explores the independent and combined effects of hepatitis B virus (HBV), hepatitis C virus (HCV) and dietary aflatoxin exposure in the etiology of HCC. Through population surveys, field trials and a series of HCC case-control studies, the patterns and natural history of HBV, HCV and aflatoxin exposures have been defined within this population. These investigations have paralleled and informed the development of molecular biomarkers of these etiologic agents and contributed to understanding the complex mechanisms involved in hepatocarcinogenesis. We discuss preventive approaches to reduce the global burden of HCC, emphasizing The Gambia Hepatitis Intervention Study, a countrywide randomized controlled trial designed to document the efficacy of HB vaccination in preventing HBV infections and HBV-related HCC. By recognizing the synergy of applying molecular techniques to population-based epidemiological studies, the portfolio of Gambian research projects presented provides a model for partnering etiologic and mechanistic investigations with applied research.

Hepatitis B immunization and hepatocellular carcinoma: The Gambia Hepatitis Intervention Study.

The Gambia Hepatitis Intervention Study (GHIS) was initiated by the International Agency for Research on Cancer (IARC) in 1986. It consisted of a randomized trial in the Gambian population, aiming to evaluate the protection provided by HBV vaccination administered during the first year of life against primary infection, the development of chronic carriage, and primary liver cancer. The results, reported at 9 years from the introduction of the vaccine as conferring a high degree of protection (83% against primary infection and 95% against chronic carriage), are briefly discussed. It is expected that HBV vaccination will result in a reduction of mortality from hepatocellular carcinoma, the most frequent cancer in males in sub-Sahara Africa.

EPI vaccines-induced antibody prevalence in 8-9 year-olds in The Gambia.

OBJECTIVES: We evaluated antibody prevalence to measles, polio 1 and 3, and tetanus toxoid antibodies in 8-9 year-old children in The Gambia within the framework of the Gambia Hepatitis Intervention Study (GHIS), a large vaccine trial aimed at evaluating vaccine efficacy against hepatitis B virus (HBV) infection, chronic carriage and primary liver cancer in a high risk population. The results of the present survey were compared with a previous survey performed with the same objectives and same methodology but in different children at 3-4 years of age. METHODS: Four clusters of 200 children each were sampled as representative of the whole country. Children would have received BCG, diphtheria-pertussis-tetanus vaccine (DPT), poliovirus vaccine (OPV), measles and yellow fever immunization. The measles haemoagglutination inhibition test (HAI) was used to detect measles antibody. Antibodies to polioviruses 1 and 3 were tested using the standard polio neutralization assay described in the EPI manual (WHO 1990). An enzyme-linked immuno-sorbent assay (ELISA) was used to measure tetanus toxoid antibodies. RESULTS: A high proportion of children were fully vaccinated in both age groups. Measles antibody concentrations were < or =1 : 8 in 8.2% of 8-9 year-old vaccinated children. In the previous survey of 3-4 year-old children this was 11.3%. In the present survey, GMC was lower than in the 3-4 year-old children; 88% of 3-4 year-olds and 89% of 8-9 year-olds had detectable antibody levels against poliovirus type 1. Fewer children at 8-9 years of age had antibodies against poliovirus type 3 than 3-4 year-olds (78%vs. 89% P < 0.001). A significant overall lower proportion of 8-9 year-old children had detectable tetanus toxoid antibodies compared to 3-4 year-old children (87%vs. 95% P < 0.001), as well as those who received four doses of DPT (90%vs. 97% P < 0.001). Conclusions High vaccine coverage is achieved in The Gambia with EPI. With time the number of vaccinated children who are not protected against measles, poliovirus 3 and tetanus increases. Besides the maintenance of high vaccine coverage in infants and young children, booster doses of some of the EPI vaccines in adolescents should be considered.

The Gambia Liver Cancer Study: Infection with hepatitis B and C and the risk of hepatocellular carcinoma in West Africa.

Hepatocellular carcinoma (HCC) is the most common cancer in The Gambia. Hepatitis B virus (HBV) infection is endemic, with 15% to 20% of the population being chronic carriers, whereas hepatitis C virus (HCV) prevalence is low. We recruited 216 incident cases of HCC and 408 controls from three sites. HBV carriage was present in 61% (129/211) of HCC patients and 16% (64/402) of controls, whereas 19% (36/191) of HCC patients were HCV seropositive compared with 3% (11/382) of controls. HCC patients with HCV were notably older and were more likely to be female than those with HBV. Increased HCC risk was strongly associated with chronic HBV (odds ratio, 16.7; 95% CI, 9.7-28.7), HCV (16.7; 6.9-40.1), and dual infection (35.3; 3.9-323). We interpret the additive nature of risk with coinfection as representative of HBV and HCV acting primarily through shared steps in the multistage process of hepatocarcinogenesis. HCV infection was not observed among younger participants, suggesting a possible cohort effect. Reasons for the striking age and gender differences in HCC associated with HBV compared with HCV are unclear, but transmission patterns and age at exposure may be factors. In conclusion, in a standardized evaluation of well-characterized study participants from The Gambia, most cases of HCC are attributable to HBV (57%), but HCV adds a significant fraction (20%), especially among older patients and females. If HCV transmission is not perpetuated in future cohorts, focusing available resources on HB vaccination efforts could greatly ameliorate a major cause of cancer death in sub-Saharan Africa.

Frequent and preferential infection of Treponema denticola, Streptococcus mitis, and Streptococcus anginosus in esophageal cancers.

Multiple cancers frequently occur in the upper digestive tract. One possible explanation is that specific bacterial infection stimulates the normal epithelium to initiate inflammation and/or promotes carcinogenesis. This study was undertaken to determine which bacterial species is predominantly associated with esophageal cancer. We examined the bacterial diversity in this type of cancer and in the saliva from healthy people by using a culture-independent molecular method. Here we report the preferential and frequent infection of the oral periodontopathic spirochete Treponema denticola (T. denticola), Streptococcus mitis (S. mitis), and Streptococus anginosus (S. anginosus) in esophageal cancer from different regions of the world, and we also describe the induction of inflammatory cytokines by infection of S. anginosus and S. mitis. Our present data suggest that these three bacteria could have significant roles in the carcinogenic process of many cases of esophageal cancer by causing inflammation and by promoting the carcinogenic process, and that eradication of these three bacteria may decrease the risk of recurrence.

Ser-249TP53 mutation in tumour and plasma DNA of hepatocellular carcinoma patients from a high incidence area in the Gambia, West Africa.

Hepatocellular carcinoma (HCC) is frequent in areas of high exposure to aflatoxin and high prevalence of HBV infection, such as western Africa and south-east China. A selective mutation in TP53 (AGG-->AGT at codon 249, Arg-->Ser) has been identified as a hotspot in HCCs from such areas, reflecting DNA damage caused by aflatoxin metabolites. Recent studies have shown that circulating free DNA can be retrieved from human plasma, and it is hypothesised that plasma DNA may serve as a source for biomarkers of tumorigenic processes. In our study, we have determined the prevalence of Ser-249 mutation, using a PCR-restriction digestion method, with selective use of short oligonucleotide mass spectrometry analysis (SOMA), in a series of 29 biopsy specimens of HCC from The Gambia in West Africa. Overall, we identified the Ser-249 mutation in 35% (10/29) of the tumours. In parallel, we tested 17 plasma samples from HCC patients with matching tumour tissue. The 249 status concordance between tumour tissues and matched plasma was 88.5%. These results indicate that the Ser-249 mutation is common in HCC in The Gambia (35%), although a higher prevalence has been reported in other regions with high population exposure to aflatoxin (e.g., eastern China: >50%). Moreover, our studies indicate that plasma is a convenient source of liver tumour-derived DNA, thus holding promise for earlier detection and diagnosis of cancer.