El carcinoma hepatocelular (CHC) es el principal cáncer primario de hígado y una de las principales causas de muerte asociada a una neoplasia. Aunque algunos de los mecanismos directos e indirectos implicados en la transformación celular han sido explorados, persisten varios interrogantes del proceso de la hepa tocarcinogénesis; entre ellas la secuencia de las etapas del proceso oncogénico y las alteraciones genéticas y epigenéticas. En particular se ha descrito la desregulación de la vía de señalización Wnt/β-catenina, como un evento decisivo en el desarrollo de diferentes tipos de cáncer. El objetivo de este estudio es determinar la frecuencia de la hipermetilación de los promotores de los genes de la vía de señalización Wnt/β-catenina, APC y E-caderina, en casos de CHC. Las muestras de estudio corresponden al banco de tejidos hepáticos embebidos en parafina de casos de CHC diagnosticados en el periodo 2000-2008, en tres hospitales de las ciudades de Medellín y Bogotá. La hipermetilación de los promotores se analizó con la técnica "Methylation Specific PCR" (MSP) y la expresión de las proteínas correspondientes por inmunohistoquímica (IHQ). La metilación de los promotores de APC y E-caderina, ha sido detectada en 4/21 (19%) y en 4/11 (36.4%) de las muestras analizadas, respectivamente. Hasta el momento se han procesado 20 muestras por IHQ, tanto para APC como para E-caderina. Según la lectura de las primeras 7 placas, APC se detectó en el 71.4% de los casos y E-caderina en el 57.1% de los casos. Los resultados preliminares, sugieren que la hipermetilación de los promotores de estos genes es un evento importante en CHC. Está en curso el análisis del total de muestras por MSP y por IHQ y confirmar los resultados de MSP por pirosecuenciación. Financiación: Colciencias y Universidad de Antioquia. 

The incidence rates of hepatocellular carcinoma (HCC) show large geographic variations, globally reflecting the prevalence of two main aetiologic factors, hepatitis B (HBV) and/or C (HCV) virus infection and exposure to high levels of aflatoxin in the diet (Chen et al. 1997). The highest incidence rates are observed in regions where most of the population is exposed to both factors, such as in parts of eastern Asia and in sub-Saharan Africa (Parkin et al. 2001). These high incidences are consistent with the fact that HBV chronicity and exposure to aflatoxin have a multiplicative effect of risk for HCC. Depending on aetiology and geographic area, mutations in TP53 show striking differences in prevalence and pattern. In Europe and the US, where alcohol is a major risk factor in addition to viral infections, mutations occur in about 25% of HCC and show as much diversity in their type and codon position as in most other epithelial cancers. However, in high incidence areas such as Mozambique, Senegal, The Gambia (Africa) and Qidong county (China), TP53 is mutated in over 50% of the cases and the vast majority of these mutations are a single missense, hotspot mutation at codon 249, AGG to AGT, resulting in the substitution of arginine into serine (249ser). This mutation is uncommon in regions where aflatoxin is not present at significant levels in the diet. In areas of intermediate exposure to aflatoxin, as for example in Thailand, the prevalence of the 249ser mutation is intermediate between high- and low-incidence areas. Thus, there is a dose-dependent relationship between exposure to aflatoxin, incidence of HCC and prevalence of 249ser mutation. Aflatoxins are toxic and carcinogenic metabolites produced by several varieties of molds, mainly Aspergillus flavus and Aspergillus parasiticum. These molds contaminate a wide range of traditional agricultural products in countries with hot, humid climates, including maize, peanuts and cottonseeds. The toxins are present at significant levels in crops at the time of harvest but their concentration further increases under poor conditions of long term food storage, in particular during the rain season. Thus, in these regions, most inhabitants of rural areas are highly exposed to aflatoxins, with seasonal variations reflecting the consumption of stored versus fresh foodstuff. Population-based surveys have demonstrated the presence of serum aflatoxin-albumin adducts in over 95% of the normal population in The Gambia, West Africa. Exposure starts in the perinatal period, through in utero transfer and breast-feeding, and continues throughout life, mainly from consumption of peanuts. Time patterns of aflatoxin-albumin adduct levels correlate with the seasonal availability of peanuts. There is strong experimental evidence that aflatoxins are potent hepatocarcinogens in rodents. In humans, there are good ecological correlations between the risk of HCC and the presence of biomarkers of aflatoxin exposure in serum or in urine .The most significant carcinogenic aflatoxin is B1 (AFB1), which is the most abundant in the diet. AFB1 is metabolized in the liver by several CYP450 enzymes (mainly 1A2 and 3A4) to a reactive AFB1-8,9- exo-epoxide (Mace et al. 1997). This metabolite generates a primary DNA adduct (8-9, dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin; AFB1-N7-Gua), naturally converted to two secondary lesions, an apurinic (AP) site and a stable, AFB1-formamidopyrimidine (AFB1-FAPY) adduct The latter is considered as the most mutagenic lesion (Smela et al. 2002). The sequence context of codon 249 (AGGCC) represents a site of intermediate affinity for the formation of AFB1-induced lesions. Other codons in TP53, including some codons that are ";hotspots"; in many cancers (codon 245, 248 and 273), have a similar or even greater affinity for AFB1 than codon 249. Thus, the selectivity for 249ser in HCC cannot be solely explained by thepreferential formation of adducts at this position and other factors must play a role to select this particular mutation as the major carcinogenic one in liver cells exposed to aflatoxins. There is evidence that imperfect DNA repair may increase the risk of mutagenesis and carcinogenesis induced by AFB1. Higher levels of AFB1-DNA adducts have been detected in the placenta of healthy women from Tawain carrying the Gln399 allele of XRCC1, an enzyme involved in base excision repair, causing slower repair and persistence of DNA adducts. Together, these results suggests that deficient DNA repair does not explain the high prevalence of 249ser in HCC, and that biological selection may play a role to facilitate the clonal expansion of cells carrying 249ser during the development of HCC.

With over 500 000 annual deaths, Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and a leading cause of death in developing countries where about 80% of the cases arise. Risk factors include chronic hepatitis infections (hepatitis B, (HBV) and hepatitis C (HCV) viruses), alcohol, dietary contaminants such as falatoxins The incidence shows important geographic variations, accor In southern Asia, HCC development is mainly related to the endemic Hepatitis B Virus (HBV) infection, cases with hot spot mutation in codon 249 (249ser) of TP53 tumor suppressor gene were also described and associated to a low-intermediate exposure rate to Aflatoxin B1 (AFB1). Presence of Hepatitis C Virus (HCV) infection was also detected in 12 - 17% of HCC cases. Despite the increasing number of studies identifying viral/host interactions in viro-induced HCC or describing potential pathways for hepatocarcinogenesis, precise mechanism has not been identified so far. HBV was demonstrated to enhance hepatocarcinogenesis by different manners; HBV chronic infection is associated to active hepatitis (CAH) and cirrhosis which are hepatic complications considered as early stage for HCC development. These complications mobilise the host immune response, the resulting inflammation initiates and selects the first genetic alteration at the origin of loss of cell control. Moreover, HBV can also promote carcinogenesis through genetic instability generated by its common integration in host DNA. HBV proteins, as HBx, was proven to interact with a variety of targets in the host cell including protein or host transcription factor such as, in particular, the p53 protein or the transcription factor E4F, which is implicated in growth, differenciation and senescence. Specific HBV mutations or distinct HBV genotypes are associated to higher risks factors for HCC or hepatic complications leading to HCC. In summary, active HBV replication potentially disrupts gene integrity, may lead to oncogenic activation through several parallel mechanism, and the role of each of these mechanism may vary with the molecular diversity of viral genotypes.

The Gambia Hepatitis Intervention Study (GHIS) is a collaborative undertaking by the International Agency for Research on Cancer, The Government of the Republic of The Gambia and the Medical Research Council of the United Kingdom. This programme was launched in 1986 with the objective of evaluating the efficacy of Hepatitis B (HB) vaccination in childhood in the prevention of HB infection, chronic liver disease and primary liver cancer in a population at high risk. The implementation of this trial involves three overlapping phases: Phase l (1986-1990): V Vaccination accination of appro approximately ximately 60,000 children. HB vaccine, which was approved by the World Health Organisation, was integrated into the Gambian Expanded Programme of Immunisation (EPI) in a phased manner over a four-year period from July 1986 to February 1990. During this period, two groups of children were recruited, one comprising about 60,000 children who received all vaccines in the EPI schedule plus the HB vaccine, the other comprising a similar number of children who received all vaccines except HB. Since February 1990, HB vaccination is offered to all newborns as part of the EPI schedule in The Gambia. Phase ll (1991-1997): Estimate of efficacy of HB vac- vaccine cine against infection and chronic carriage. Longitudinal and cross-sectional surveys were carried out in selected groups of vaccinated (Group 1) and unvaccinated (Group 2). These two subsets have provided evidence of the short-term efficacy of HB vaccine in preventing infection and chronic carriage. By the end of the first decade of life, the vaccine prevents 84% and 94% of HBV infections and chronic carriage, respectively, despite waning antibody levels during the period. Phase lll (since 1998): Long-term follow-up through Cancer Registration. The aim of this phase is to carry out a surveillance of the population of The Gambia, to identify cases of chronic liver disease (cirrhosis) and liver cancer. A linkage is made between the records of cases occurring in subjects within the age-range of the GHIS cohort, and the GHIS database of vaccinated children, to determine whether the individual belongs to the vaccinated or unvaccinated cohort. The components of Phase III are: 1. Detection and ascertainment of cancer cases and cases of chronic liver disease in the population of The Gambia, through support to liver cancer diagnosis in the public and private health sector, and support to Laboratory and Histopathology Services. 2. Registration of cancer cases and of cases of chronic liver disease through the National Cancer Registry (NCR), a population-based cancer registry established in 1986. 3. Record linkage of identified cases with the GHIS database of vaccinated/non-vaccinated children, so that the net effect of HB vaccination in preventing liver cancer can ultimately be assessed. In parallel with the development of the three phases above, the GHIS framework has fostered studies on viral, environmental and genetic factors in hepatocellular carcinoma, biomarkers of HB Infection and aflatoxin exposure, long term efficacy of HB vaccination and monitoring of breakthrough injections.

The tumor suppressor gene TP53 is frequently inactivated by gene mutations in many types of human sporadic cancers, and inherited TP53 mutations predispose to a wide spectrum of early-onset tumors (Li-Fraumeni et Li-Fraumenilike Syndromes). All TP53 gene variations (somatic and germline mutations, as well as polymorphisms) that are reported in the scientific literature or in SNP databases are compiled in the IARC TP53 Database. This database provides structured data and analysis tools to study mutation patterns in human cancers and cell-lines and to investigate the clinical impact of mutations. It contains annotations related to the clinical and pathological characteristics of tumors, as well as the demographics and carcinogen exposure of patients. The IARC TP53 web site (http://www-p53.iarc.fr/ ) provides a search interface for the core database and includes a comprehensive user guide, a slideshow on TP53 mutations in human cancer, protocols and references for sequencing TP53 gene, and links to relevant publications and bioinformatics databases. The database interface allows download of entire data sets and propose various tools for the selection, analysis and downloads of specific sets of data according to user's query. Recently, new annotations on the functional properties of mutant p53 proteins have been integrated in this database. Indeed, the most frequent TP53 alterations observed in cancers (75%) are missense mutations that result in the production of a mutant protein that differ from the wildtype by one single amino-acid. The characterization of the biological activities of these mutant proteins is thus very important. Over the last ten years, a great amount of systematic data has been generated from experimental assays performed in yeast and human cells to measure the impact of these mutations on various protein properties: (1) transactivation activities (TA) of mutant proteins on reporter genes placed under the control of various p53 responseelements, (2) capacity of mutant proteins to induce cellcycle arrest or apoptosis, (3) ability to exert dominantnegative effect (DNE) over the wild-type protein, (4) activities of mutant proteins that are independent and unrelated to the wild-type protein (gain of function, GOF). Prediction models based on interspecies protein sequence conservation have also been developed to predict the functional impact of all possible single amino-acid substitutions. These data have been used to produce systematic functional classifications of mutant proteins and these classifications have been integrated in the IARC TP53 database. New tools have been implemented to visualize these data and analyze mutation frequencies in relation to their functional impact and intrinsic nucleotide substitution rates. Thus, the database allows systematic analyses of the factors that shape the patterns and influence the phenotype of missense mutations in human cancers. In a recent analysis of the database, we showed that that loss of TA capacity is a key factor for the selection of missense mutations, and that difference in mutation frequencies is closely related to nucleotide substitution rates along TP53 coding sequence. TA capacity of inherited missense mutations was also found to be related the age at onset of specific tumor types, mutations with total loss of TA being associated with earlier cancer onset cancers compared to mutations that retain partial trans-activation capacity. Furthermore, 80% of the most common mutants show a capacity to exert dominant-negative effect (DNE) over wildtype p53, compared to only 45% of the less frequent mutants studied, suggesting that DNE may play a role in shaping mutation patterns. Hotspot mutations have been linked to exposure to environmental factors in several cancers: tobacco smoke in lung cancer, tobacco smoke and alcohol in head and neck cancers, aromatic-amines in bladder cancer, aflatoxine-B1 and HBV in liver cancer, and UV in skin cancer. In lung cancers, four specific mutants are observed at high frequencies, V157F, R158L, R248L and R273L. These hotspots are due to G>T transversions that have been shown to be caused by the presence of benzo(a)-pyrene diol epoxide (BPDE) adducts on guanines at these codons. BPDE is the main metabolite of benzo(a)-pyrene, one of the most potent carcinogens present in high quantity in tobacco smoke. In hepatocellular carcinomas from less developed regions, one specific mutant is observed at a high frequency, R249S. Although the precise mechanism remain unknown, its presence is attributed to an interaction between HBV infection and mutagenesis by the food contaminant carcinogen aflatoxine-B1. In these examples, all hotspot mutants are defective for TA. These observations show how mutagenesis by environmental carcinogens and selection of loss of trans-activation can shape TP53 mutation patterns. In conclusion, the integration of standardized annotations on the functional impact of missense mutations in the IARC TP53 database provides a powerful framework for the analysis of ";functional"; patterns of mutations in cancers, the detection of genotype/phenotype associations and provide new insights into the factors that shape mutation patterns and influence mutation phenotype, which may have clinical interest.   REFERENCES 1. Petitjean A, Mathe E, Kato S, Ishioka C, Tavtigian SV, Hainaut P, Olivier M. Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database. Hum Mutat. 2007 Feb 20;28(6):622-629. 2. Petitjean A, Achatz MI, Borresen-Dale AL, Hainaut P, Olivier M. TP53 mutations in human cancers: functional selection and impact on cancer prognosis and outcomes. Oncogene. 2007 Apr 2;26(15):2157-65.

HBV and HCV play key roles in the etiopathogenesis of Hepatocellular carcinoma (HCC) . Studies mostly based on cases from Western countries suggest distinct genetic pathways of carcinogenesis involving either TP53 or CTTNB1 mutations. Inappropriate reactivation of Wnt pathway due to mutations in CTNNB1 (Beta-Catenin) gene itself is also frequently reported. Mutant Beta-catenin escapes to ubiquitination and down regulation by GSK3-B, it accumulates and trans-activates variety of oncogenes involved in neoplasmic transformation mimicking Wnt pathway activation. Taking into consideration viral infection, chromosome instability and TP53 /Beta-catenin alterations, Laurent-Puig et al. described two distinct HCC profiles in a serie of 137 HCC cases , the first one associates HBV infection with frequent chromosomal alteration and distributes with TP53 mutations, the second would be observed in HBV negative large sized tumors and distributes with Beta-catenin mutations. We have investigated the status of HBV and HCV infections and of genetic alterations in TP53 and CTTNB1 in 26 patients with HCC from Thailand. In tumours, HBV DNA was found in 19 cases (73%) and HCV RNA in 4 cases (15.4% cases), 3 of whom were co-infected. Among the 19 HBV positive cases, sequencing of S gene showed genotype C in 82% and genotype B in 18%. Furthermore, 5/19 cases were negative for HBsAg and were categorized as occult HBV infections. TP53 mutations were detected in 9 cases (34,6%) including 7 mutations at codon 249 (AGG to AGT, arginine to serine), considered as ";fingerprint"; of mutagenesis by aflatoxin metabolites. All cases with 249ser mutation had overt HBV infection. CTNNB1 mutations were found in 6/26 cases (23%), 4 of whom also had TP53 mutation. There was no significant association between CTTNB1 mutations and viral infection status. These results suggest that mutagenesis by aflatoxin may have an impact greater than recognized sofar in the etiopathogenesis of HCC in Thailand. Furthermore, TP53 and CTNNB1 mutations do not appear as mutually exclusive, and TP53 249ser mutation is associated with overt HBV infection. Thus, HCC in this context may develop according to a sequence of genetic events that includes both TP53 and CTNNB1 mutations.