The 249Ser mutation was found in 28% of HCC samples included in this study, it is lower somewhat similar to our previous finding of immunohistochemistry reactivity for p53 in 35% of Brazilian HCC cases from our group . At that time only immunoexpression of this protein was analyzed, not the determination of the mutation at the hotspot(s) responsible for this over expression.
The 28% rate of 249Ser TP53 point mutation found is rather high, a finding probably related to previous studies of contamination of Brazilian food with aflatoxin [27, 1]. AFB1 contamination is a public health problem in Brazil. It is also important to remind that codon 249 is the responsible for 33% of TP53 mutations in HCC, but it is not the only TP53 hotspot. In a search on IARC database, it is possible to find other TP53 hotspots. It is described other 143 mutations besides 249Ser in HCC. Other common mutation takes place in codons 175, 245, 248 273, and 282. The other codons have an individual contribution to TP53 mutation of less than 2% . This information can justify the difference between the frequency of the mutation found in this study and the higher frequency of over expressed p53 described previously .
Our results are strikingly differing to those published in European countries where, not only 249Ser, but also all TP53 hotspots do not have relevant influence in HCC carcinogenesis. Kubicka  showed that none of his 20 HCC samples had the 249Ser point mutation and between all cases only one had p53 over expression due to a 248 codon mutation. USA had a similar result: even though there was 5/23 cases of HCC with p53 over expression all of them were wild type for the 249 codon .
It was described a 45% frequency of p53 over expression on his samples – a high rate for Europeans samples – even though no codon 249 mutation was found .
On the other hand, in countries with high incidence of HCC, the 249Ser point mutation has an important role in the liver carcinogenesis. In Senegal this kind of mutation can be detected in 67% of HCC patient . In regions like Gambia (Africa) and Qidong (China) this mutation frequency can reach up to 50%. The mutation rate found in our study is much higher comparing to European countries yet lower than in regions where aflatoxin exposure is endemic. Unfortunately, there are not many studies about 249Ser frequency in Latin-American countries. However, in Mexico it was described a 3/16 (19%) frequency of this mutation in HCC . This 249Ser frequency may be related to the fact that Mexico is one of the biggest corn consumers in the world (almost 120 kg per capita per year). This fact associated with an imperfect foodstuff storage conditions and manipulation may result in a large AFB1 intake. Another fact which should be discussed is the fact that up to 2005, Mexican legislation about Aflatoxin contamination lack of rules about Aflatoxin presence in milk .
Although, AFB1 contamination has been controlled in Brazil, there are still cases of foods presenting AFB1 levels beyond the tolerated level, for example: 20 kinds of peanuts were interdicted during February 2005 and July 2006 by ANVISA (National Sanitary Administration Agency) . The maximum AFB1 level in food allowed by ANVISA was 30 ppb up to 2002 and 20 ppb after that. FDA has always recommended an AFB1 level less than 15 ppb. European Food Safety Authority (EFSA) allows only 0.1 ppb of AFB1 in some cases and there are specific rules about food for child nutrition . Moreover, residual effects of exposition to higher aflatoxin levels before 2002 will probably yield high rates of AFB-related HCC in Brazil for the next few decades.
Our data might suggest that 249Ser is related not only to poorly differentiated HCC but also to also larger tumors. It was showed that mutations among TP53 are associated with poor differentiation level [37, 38]. However, in both studies there was a low 249Ser frequency and the poor differentiation was associated only to TP53 mutation and not to 249Ser itself. Moreover, neither of those studies found association between TP53 mutation and tumor size. Age and vascular invasion did not present correlation with the specific mutation. Despite not presenting statistical relationship with 249Ser, the mutation had a tendency of being more frequent in cirrhotic liver. The same can be said about gender: The mutation was observed slightly more frequently in males rather in female ones (p = 0.076).
The fact that the mutation was more frequent among larger and less differentiated tumors could suggest that 249Ser is a late event on liver carcinogenesis, which may sound, be against the hypothesis that AFB1 is a causal agent of HCC. However, there are studies that describe the selective advantages that a liver cell carrying this mutation may have. Among them it could be pointed: enhancement of cell growth , inhibition of wild-type p53 mediated apoptosis  and finally p53 249ser has great efficiency in suppressing wild-type p53 activity .
Interestingly, all HCC samples had low levels of mutant p53-249 ser mutation as compared to wild-type p53 DNA. Because larger and less differentiated tumors are less homogeneous it is possible that only part of tumor displayed the p53-249ser mutation, following the suggesting that 249ser is a late event on liver carcinogenesis. Alteration in p53 may be important event in the transformation of hepatocytes of regenerative nodules in a damaged organ (the "field effect") to the malignant phenotype. Also it is possible that part of non-tumor tissue with wild-type p53DNA contributed for this situation, since it was not performed micro dissection to separated only tumor cells. Nevertheless, this situation did not preclude the detection p53 249ser mutation in a significative number of tumors, suggesting a role of aflatoxin in the carcinogenesis of liver tumor in our country.
Statistical relationship between p53 249ser mutation and HBV presence in the hepatocytes was not found. This, however, may be due to the fact that only HBV DNA presence was analyzed and not its gene expression. Many studies describe that the pathway, which leads to the 249ser mutation, is related with HBx expression however it was not this project the aim. In this study HBV DNA presence was not found being related to neither cirrhosis status nor vascular invasion of the tumor.