Mutation of p53 gene in hepatocellular carcinoma in Taiwan.

To elucidate the role of p53 mutation in hepatocarcinogenesis in Taiwan, a hepatitis B viral infection hyperendemic area, exons 5 to 8 of the p53 gene in the tumor tissue of 61 hepatocellular carcinomas were amplified and sequenced. A total of 20 cases (32.8%) were found to have mutations; 36.6% (15 of 41) for the hepatitis B surface antigen positive group and 25.0% (5 of 20) for the hepatitis B surface antigen negative group. The corresponding normal liver showed no mutation. The mutation is widely distributed throughout exons 5 to 8. Only 4 cases (6.6%), all positive for hepatitis B surface antigen, had a specific hot spot mutation at codon 249 with G to T transversion. Our results show that scattered point mutations in p53 are not uncommon in hepatocellular carcinoma samples from Taiwan and may be important in the development of this cancer. However, the aflatoxin related specific mutation seems much less related to the genesis of hepatocellular carcinoma in Taiwan.     

Hepatitis B, aflatoxin B(1), and p53 codon 249 mutation in hepatocellular carcinomas from Guangxi, People's Republic of China, and a meta-analysis of existing studies.

The incidence of hepatocellular carcinomas (HCC) varies widely worldwide, with some of the highest incidence rates found in China. Chronic infection with the hepatitis B virus (HBV) and exposure to aflatoxins in foodstuffs are the main risk factors. A G to T transversion at codon 249 of the p53 gene (249(ser)) is commonly found in HCCs from patients in regions with dietary aflatoxin exposure. Because HBV infection is often endemic in high aflatoxin exposure areas, it is still unclear whether HBV acts as a confounder or as a synergistic partner in the development of the 249(ser) p53 mutation. Our report has two aims. First, we contribute data on HCCs from southern Guangxi, a high aflatoxin exposure area. Using DNA sequencing, we found that 36% (18 of 50) of tumors had a 249(ser) mutation. Also, 50% (30 of 60) were positive for p53 protein accumulation and 78% (28 of 36) were positive for HBV surface antigen, as detected by immunohistochemistry. Second, we present a meta-analysis, using our results along with those from 48 published studies, that examines the interrelationships among aflatoxin exposure, HBV infection, and p53 mutations in HCCs. We used a method that takes into account both within-study and study-to-study variability and found that the mean proportion of HCCs with the 249(ser) mutation was positively correlated with aflatoxin exposure (P = 0.0001). We found little evidence for an HBV-aflatoxin interaction modulating the presence of the p53 249(ser) mutation or any type of p53 mutation.     

Absence of p53 mutation at codon 249 in duck hepatocellular carcinomas from the high incidence area of Qidong (China)

Dietary aflatoxin and hepatitis B virus infection may play arole in generating the p53 tumor suppressor gene codon 249 hotspotmutation found in human hepatocellular carcinomas (HCCs) fromQidong (China) and southern Africa. No data are available onthe HCC site-specific mutation of the p53 gene in hepadnavirus-infectedanimals exposed to AFB₁. We have searched for the presence ofp53 gene codon 249 mutations in both duck hepatitis B virus(DHBV) positive and negative HCCs of domestic ducks from Qidong,where the human p53 hotspot is so prevalent, as well as in duckHCCs experimentally induced by AFB₁. Direct sequencing of DNAamplification products encompassing p53 codon 249 did not revealany mutations in 11 HCCs from Qidongducks, regardless of thestatus of DHBV infection. In addition no mutation was detectedin four HCCs from AFB₁-treated ducks. This contrasts with thehuman data; however, in humans, the mutation and the preferentialbinding of AFB₁ to codon 249 occurs at the third nucleotideG, while in duck, the codon 249 lacks this G residue. The DNAsequence of adjacent codons is also different in the two specieseven though the amino acid sequence is identical. This may explainthe low frequency of mutation we have observed. In addition,species differences in metabolism and DNA repair could influencethe occurrence of codon 249 mutations.     

Genetic alterations in hepatocellular carcinomas: association between loss of chromosome 4q and p53 gene mutations.

The major risk factors for hepatocellular carcinomas (HCC) in high incidence areas include infection with hepatitis B and C viruses (HBV, HCV) and exposure to aflatoxin. Genetic alterations in 24 liver resection specimens from Shanghai and Qidong were studied. Hepatitis B virus was integrated in all patient samples, and a null phenotype for the GSTM1 enzyme was present in 63% of patients. Alteration of p53 was present in 95% (23/24) of cases: mutations of the p53 gene in 12 HCC, p53 overexpression in 13 and loss of heterozygosity (LOH) of chromosome 17p in 17. All seven HCCs with a p53 mutation from Qidong and three of five from Shanghai had the aflatoxin-associated point mutation with a G to T transversion at codon 249, position 3. No HCC had microsatellite instability. LOH of chromosome 4q, 1p, 16q and 13q was present in 50%, 46%, 42% and 38%, respectively, and 4q was preferentially lost in HCCs containing a p53 mutation: LOH of 4q was present in 75% (9/12) of HCC with, but only 25% (3/12) of HCC without, a p53 gene mutation (P = 0.01). These data indicate a possible interaction between p53 gene mutation and 4q loss in the pathogenesis of HCC.     

Different frequencies of p53 codon-249 hot-spot mutations in hepatocellular carcinomas in Jiang-su province of China.

Environmental carcinogens often induce specific mutations in the p53 gene, apparent in tumors. The relation between aflatoxin B1 (AFB1 )-related hepatocellular carcinomas (HCCs) and hot spot at codon 249 of the p53 gene has received a great deal of attention, but its significance is still controversial. To clarify this problem, we analyzed the p53-mutational status of HCCs in Jiang-su province in China, where AFB1 contamination of the staple food significantly differs between the northern and southern parts (prominent only in the latter), while other conditions are quite similar. Background liver status and mutations in exons 5 to 8 of p53 in a total of 31 cases were divided approximately equally between the 2 areas. In all, 15 tumors exhibited a total of 17 mutations in the p53 gene; 9 cases from the southern part of the province had the hot-spot mutation at codon 249 (9/16, 56%), but only one case from the northern part (1/15, 8%). These results suggest that AFB1 contamination may correlate with codon-249 mutations in HCC.     

The chemistry and biology of aflatoxin B(1): from mutational spectrometry to carcinogenesis

Dietary exposure to aflatoxin B(1) (AFB(1)) is associated with an increased incidence of hepatocellular carcinoma (HCC), especially in populations in which exposure to hepatitis B virus (HBV) is a common occurrence. Most HCC samples from people living where HBV is prevalent have one striking mutational hotspot: a GC-->TA transversion at the third position of codon 249 of the p53 gene. In this review, the chemical reaction of an electrophilic derivative of aflatoxin with specific DNA sequences is examined, along with the types of mutations caused by AFB(1) and the sequence context dependence of those mutations. An attempt is made to assign the source of these mutations to specific chemical forms of AFB(1)-DNA damage. In addition, epidemiological and experimental data are examined regarding the synergistic effects of AFB(1) and HBV on HCC formation and the predominance of one hotspot GC-->TA transversion in the p53 gene of affected individuals.     

乙肝病毒/黄曲霉毒素B1双暴露相关性肝细胞癌的p53基因249位点突变与p53蛋白表达的关系

目的研究乙肝病毒/黄曲霉毒素B1双暴露相关性肝细胞癌的p53基因249位点突变与p53蛋白表达关系。方法通过IHG特殊免疫组织化学法检测55例手术切除经病理证实为原发性肝癌的HCC组织及10例正常肝组织中AFB1-DNA加合物的暴露情况,并根据是否同时存在HBV暴露加以分组,通过免疫组化S-P法检测并比较各组间p53蛋白的表达情况。同时,通过PCR结合直接测序的方法检测其p53基因第7外显子249密码子的突变情况。结果 p53基因第7外显子249位点的突变在实验组A与对照组C中均具有较高阳性突变率,其突变率分别为68.75%（22/32）、63.64%（7/11）,在对照组B中的突变率较低,为16.67%（2/12）,在正常对照组中无1例出现有突变。其中实验组A与对照组B及正常对照组D的比较差异具有统计学意义（P〈0.05）,而与对照组C比较差异无统计学意义（P〉0.05）;p53蛋白在其基因249位点突变阳性组与阴性组中的表达阳性率分别为92.86%（26/28）、60.87%（16/27）,差异具有统计学意义（P〈0.05）。结论 HCC的发生过程中,AFB1暴露与p53第7外显子249位点的突变密切关系相关,当同时存在乙肝病毒暴露的协同作用的情况下突变率更高;p53基因的突变可能是造成HCC中p53蛋白高表达的重要因素之一。     

P53 tumor suppressor gene mutations in hepatocellular carcinoma patients in India

BACKGROUND: Specific mutations of the p53 tumor suppressor gene in hepatocellular carcinoma (HCC) have been reported from several parts of the world, but to the authors' knowledge to date the status of this gene has not been studied in HCC patients in India, where HCC is one of the major cancers and the frequency of chronic hepatitis B virus (HBV) as well as hepatitis C virus (HCV) infection and exposure to dietary aflatoxin B(1) is very high. The most frequent mutation of the p53 gene in HCC is an AGG(Arg) to AGT(Ser) missense mutation at codon 249 of exon 7. METHODS: Liver biopsy specimens from 21 HCC patients and 10 healthy controls were obtained through surgery or by needle biopsy technique. Phenol-chloroform-extracted DNA specimens were employed for the detection of HBV infection and p53 gene mutations. Nucleotide mutations of exons 4-9 of the p53 gene were analyzed by polymerase chain reaction (PCR), single strand confirmation polymorphism, and direct sequencing. Third-generation sandwich enzyme-linked immunosorbent assay (ELISA) was used for the serologic detection of HBV and HCV infection. RESULTS: Analysis of exons 4-9 of the p53 gene revealed only 3 mutations (3 of 21 specimens, 14.28%; 95% confidence interval, -0.7-29.3), 2 mutations at codon 249 showing G-->T transversions, and 1 mutation (4.7%) at codon 250 with a C-->T transition. The base substitutions at the third base of codon 249 resulted in a missense mutation leading to a change in amino acid from arginine to serine whereas at codon 250 it caused a change from proline to serine. Dot blot hybridization and PCR for HBV DNA from HCCs revealed 58.8% (10 of 17 specimens) and 90. 47% (19 of 21 specimens), positivity, respectively. ELISA for hepatitis B virus surface antigen in serum showed a positivity of 71. 42% (15 of 21 specimens), but there was only 40% positivity (8 of 20 specimens) for hepatitis B virus envelope antigen whereas 6 of 17 patients (35.29%) showed the presence of antibodies against hepatitis B virus envelope protein. No patient was found to be positive for the HCV antibody. CONCLUSIONS: The very low frequency of p53 mutations and the extremely high frequency of HBV infection (> 90%) in HCC indicate that the mutations in the p53 gene frequently found in HCC reported from different endemic areas of the world may not play a direct role in the development of HCC in India. HBV infection and, possibly, exposure to the dietary aflatoxin B(1) appear to play major roles in the molecular pathogenesis of HCC in India.     

Specific p53 mutations detected in plasma and tumors of hepatocellular carcinoma patients by electrospray ionization mass spectrometry

Hepatocellular carcinoma (HCC), a common cause of cancer deaths worldwide, has several major etiological risk factors, including infection with the hepatitis viruses and exposure to aflatoxin B1. A specific missense mutation resulting from a guanine to thymine transversion at the third position of codon 249 in the p53 tumor suppressor gene has been reported in 10-70% of HCCs from areas of high dietary exposure to aflatoxin B1. Short oligonucleotide mass analysis was compared with DNA sequencing in 25 HCC samples for specific p53 mutations. Mutations were detected in 10 samples by short oligonucleotide mass analysis in agreement with DNA sequencing. Analysis of another 20 plasma and tumor pairs showed 11 tumors containing the specific mutation, and this change was detected in six of the paired plasma samples. Four of the plasma samples had detectable levels of the mutation; however, the tumors were negative, suggesting possible multiple independent HCCs. Ten plasma samples from healthy individuals were all negative. This molecular diagnostic technique has implications for prevention trials and for the early diagnosis of HCC.     

Allele-specific PCR analysis of p53 codon 249 AGT transversion in liver tissues from patients with viral hepatitis

AGG to AGT mutations in codon 249 of the p53 tumor-suppressor gene are frequently observed in hepatocellular carcinomas (HCC) from areas where exposure to aflatoxin B₁ (AFB) occurs. We developed a sensitive allele-specific polymerase chain reaction (AS-PCR) assay to detect this point mutation in non-neoplastic human liver tissues. Three oligonucleotide primers, 1 specific for the mutant allele and 2 specific for the wild-type allele were used. The mutant allele primer differed from the wild-type allele due to a G-to-T transversion in its terminal 3′ nucleotide. The first stage involved amplification of exon 7 of p53 followed by a selective amplification of mutant codon 249 sequences. This method allowed for the detection of a mutant codon 249 allele in the presence of as many as 10⁵ copies of the wild-type allele and was 100-fold more sensitive than the restriction fragment length polymorphism-PCR technique. We have applied this AS-PCR protocol to examine codon 249 AGT transversion in tumor and matched non-tumor liver samples from North American patients with hepatitis and from Mozambiquan patients exposed to AFB. Mutations were detected in 5 of 6 samples of non-neoplastic liver from Mozambican patients, all of whom were HBsAg- or HBcAg-positive and AFB-exposed. In contrast, no mutations were detected in non-neoplastic liver from North American patients with either HBV- or HCV-derived hepatitis and cirrhosis. This procedure is a simple and powerful approach for screening p53 codon 249 AGT mutation in heterogeneous non-neoplastic hepatocyte populations. © 1996 Wiley-Liss, Inc.     

Novel adenine adducts, N7-guanine-AFB1 adducts, and p53 mutations in patients with schistosomiasis and aflatoxin exposure

INTRODUCTION: The most frequent mutation in human hepatocellular carcinoma (HCC) in populations exposed to a high dietary intake of aflatoxin B1 (AFB1) is a mutation in codon 249 of the p53 gene. Schistosomiasis is known to cause p53 mutation. We hypothesized that the combination of schistosomiasis and aflatoxin B1 increases the incidence of p53 gene mutation. METHODS: Liver tissue from 21 patients with schistosomiasis and 5 patients without schistosomiasis were analyzed for occurrence of mutations of the p53 gene and levels of N7-guanine-AFB1 adducts. RESULTS: The presence of mutations in codon 249 of p53 gene was higher in patients infected with Schistosoma haematobium (S. haematobium) than in those infected with Schistosoma mansoni (S. mansoni) or a combination of both strains (p < 0.01), compared to control subjects. No mutations were detected in p53 gene in liver DNA from schistosomiasis-free patients. Significant amounts of N7-guanine-AFB1 adducts and novel adenine-adducts (p < 0.01) were detected in patients with schistosomiasis, mostly in patients infected with S. haematobium or a combination of both strains, compared to control subjects. Conclusion: These data suggest that schistosomiasis and exposure to aflatoxin B1 act synergistically to increase the incidence of p53 gene mutation. The increase in p53 mutations may enhance progression of HCC at an early age in patients with schistosomiasis.     

p53 Mutations in human hepatocellular carcinomas from Germany.

Mutations in the p53 gene are frequent genetic alterations in human hepatocellular carcinomas. We have examined 13 cases of human hepatocellular carcinomas from Germany for the presence of p53 aberrations in exons 4 to 8 of the gene by single-strand conformation polymorphism and restriction fragment-length polymorphism analyses and by sequencing of polymerase chain reaction products. Single base substitutions occurred in two human hepatocellular carcinomas: a C:G----T:A transition at a CpG site in codon 257, and a T:A----A:T transversion at codon 273. One of these point-mutated tumors and two additional tumors without point mutations demonstrated a loss of one p53 allele. None of the tumors was mutated in codons 12 or 61 of the c-Ha-ras gene.     

Aberrations of p53 gene in human hepatocellular carcinoma from China

Allele losses and mutations have been examined in 38 cases ofprimary hepatocellular carcinomas (HCC) from different geographicareas of China by Southern, single-strand conformational polymorphism(SSCP) and direct DNA sequencing analyses. Two of 12 samplesfrom Qi-Dong and six of 18 HCCs from Shanghai showed loss ofheterozygosity (LOH) at the loci on chromosome 17p13.3. Allof the nine mutations in the p53 gene detected in HCC from Qi-Dongwere clustered at the third base of codon 249, i.e. G:C to T:A,leading to an arginine to serine change. In contrast, 18 HCCsamples from Shanghai contained three mutations at codons 249,255 and 279. These results suggested a relationship betweenthe spectrum of p53 aberration and environmental risk factorsin these two geographic areas. Since no correlation betweenthe state of HBV DNA and p53 abberration was observed, otherfactors such as dietary exposure to aflatoxin B1 (AFB1) mightbe responsible for the mutational hotspot at codon 249 in HCCsfrom Qi-Dong area.     

Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens

In lung and liver cancers, p53 mutations are mostly G:C to T:A transversions. This type of mutation is known to be induced by benzo(a)pyrene and aflatoxin B1 which are associated with the etiology of lung and liver cancers, respectively. Using a novel assay based on DNA polymerase fingerprint analysis, we identified p53 nucleotides targeted by these carcinogens. Thirteen of 14 nucleotide residues of the p53 gene which underwent G:C to T:A mutations in lung cancers were targeted by benzo(a)pyrene. Similarly, aflatoxin B1 formed adducts at a mutational hotspot specific for liver cancer. The same nucleotide (third base of codon 249), which mutates rarely in lung cancers, was not a target for benzo(a)pyrene. These in vitro observations indicate that p53 mutational hotspots identified in different tumors are selected targets specifically for the etiologically defined environmental carcinogens.     

Absence of TP53 codon 249 mutations in young Guinean children with high aflatoxin exposure.

Infection with hepatitis viruses and chronic exposure to high levels of dietary aflatoxins are the major etiologic agents for hepatocellular carcinoma in west Africa. A challenge for the prevention of hepatocellular carcinoma in this region is that both hepatitis B virus and aflatoxin exposures start early in life; indeed, aflatoxin exposures can start in utero and continue unabated throughout childhood. A mutation in the TP53 tumor suppressor gene at codon 249 (TP53 Ser249 mutation) has been reported previously for hepatocellular carcinoma tumors and matched plasma DNA samples in individuals from areas with high aflatoxin exposure. We examined whether the TP53 Ser249 mutation could be observed in DNA found in plasma of young children (ages 2-5 years) from Guinea, west Africa, a region of high aflatoxin exposure. Plasma aflatoxin-albumin adducts were present in 119 of 124 (96%) of the children, geometric mean of positives 9.9 pg/mg albumin (95% confidence interval, 8.8-11.0 pg/mg). This is the level and prevalence of exposure observed previously in adults. Following PCR amplification of plasma-derived DNA and detection using mass spectrometry, none of the samples were found to contain the TP53 Ser249 mutation. Because approximately 50% of the hepatocellular carcinomas in adults in west Africa have this specific TP53 Ser249 mutation, a lack of detection in samples from children ages <5 years may indicate that a window of opportunity for intervention exists that could be exploited to lower hepatocellular carcinoma risk.     

p53 mutations and aflatoxin B1 exposure in hepatocellular carcinoma patients from thailand

The prevalence and type of mutations in the p53 tumoursuppressor gene have been determined in 15 hepatocellular carcinomas (HCC) originating from Thailand. Direct sequencing of exons 5-8 revealed 2 mutations, an AGG to AGT (Arg → Ser) transversion at codon 249, and an ATC → AAC (lle → Asn) transversion at codon 254. Samples from the Thai patients were analyzed for the presence of aflatoxin-liver DNA and aflatoxinserum albumin adducts, and all but one were found negative. All the patients were genotyped for glutathione-S-transferase (GST) μ, an enzyme possibly involved in the detoxification of AFB₁, and 12 out of 15 had the null genotype. In general, the level of aflatoxin-albumin adducts in sera and the prevalence of p53 mutation at codon 249 in HCC were lower than in other areas at high risk of HCC, including southern China and parts of Africa.     

Hepatitis B Virus Genetic Variation and TP53 R249S Mutation in Patients with Hepatocellular Carcinoma in Thailand

Chronic hepatitis B virus (HBV) infection and dietary exposure to aflatoxin B1 (AFB1) are major risk factorsfor hepatocellular carcinoma (HCC). The aim of this study was to evaluate the role of HBV genetic variationand the R249S mutation of the p53 gene, a marker of AFB1-induced HCC, in Thai patients chronically infectedwith HBV. Sixty-five patients with and 89 patients without HCC were included. Viral mutations and R249Smutation were characterized by direct sequencing and restriction fragment length polymorphism (RFLP)in serum samples, respectively. The prevalences of T1753C/A/G and A1762T/G1764A mutations in the basalcore promotor (BCP) region were significantly higher in the HCC group compared to the non-HCC group.R249S mutation was detected in 6.2% and 3.4% of the HCC and non-HCC groups, respectively, which was notsignificantly different. By multiple logistic regression analysis, the presence of A1762T/G1764A mutations wasindependently associated with the risk of HCC in Thai patients.     

Polymorphism of XRCC1 and the frequency of mutation in codon 249 of the p53 gene in hepatocellular carcinoma among Guangxi population, China

In hepatocellular carcinoma (HCC), hotspot mutation in codon 249 of the p53 gene has been associated with exposure to aflatoxin B1 (AFB1). While the polymorphism of DNA repair gene X-ray repair cross-complementary group 1 (XRCC1) Arg399Gln may be related with AFB1-DNA adducts and gene mutations. Five hundred one HCCs were included in this study to investigate the role of the XRCC1 codon 399 polymorphism on hotspot mutation in codon 249 of the p53 gene. The genotypes of XRCC1 codon 399 and p53 codon 249 were examined by PCR-RFLP. The HCC patients with XRCC1 genotypes with 399 Gln (namely: XRCC1-AG/GG) exhibited a significantly higher frequency of the p53 hotspot mutations in codon 249 than those with the wild-type homozygote of XRCC1 [namely: XRCC1-AA, adjusted odds ratio (OR) = 6.77, 95% confidence interval (CI) = 4.34-10.57]. Compared with those individuals who did express XRCC1-AA as reference (OR = 1), moreover, individuals featuring XRCC1-AG/GG and AFB1-DNA adducts did experience a significantly greater frequency of the hotspot mutation in codon 249 of the p53 gene (adjusted OR = 28.37, 95% CI = 13.19-61.02, P < 0.01). This study suggests that the XRCC1 Arg399Gln polymorphism and AFB1-DNA adducts are associated with the increased frequency of the p53 mutations in codon 249.     

Beta-catenin mutations and expression, 249serine p53 tumor suppressor gene mutation, and hepatitis B virus infection in southern African Blacks with hepatocellular carcinoma

BACKGROUND AND OBJECTIVES: To ascertain the prevalence of deregulating mutations of beta-catenin gene, and to correlate this with the occurrence of 249(serine) p53 gene mutation and hepatitis B virus infection in southern African Blacks with hepatocellular carcinoma. METHODS: Paired cancer/non-cancerous liver tissues from 21 and cancer tissues alone from 20 Black Africans with hepatocellular carcinoma were studied. RT-PCR-SSCP and sequencing were used to detect mutations in exon 3 of the beta-catenin gene, and PCR, restriction endonuclease analysis, and sequencing to detect the p53 gene mutation. Immunostaining was used to identify beta-catenin protein expression in hepatocytes. RESULTS: No mutations in exon 3 of the beta-catenin gene were found in tumor or non-tumorous tissues. Immunohistochemical staining showed beta-catenin protein expression in membranes and cytoplasm of hepatocytes but not in the nuclei. The 249serine p53 gene mutation was detected in 27.2% of the hepatocellular carcinoma tissues but not in non-cancerous tissues. No correlation was found between beta-catenin mutation and over-expression and 249serine p53 gene mutations or hepatitis B virus surface antigenemia. CONCLUSIONS: Unlike hepatocellular carcinomas in China, Japan, and Europe, deregulating beta-catenin gene mutations do not appear to occur in southern African Blacks with this tumor and do not therefore interact with either the 249serine p53 gene mutation or hepatitis B virus infection in its pathogenesis.