肝细胞癌组织和癌周组织中p53基因密码子249突变的比较研究

目的：本研究通过比较研究ＨＣＣ 癌组织和癌周组织中ｐ５３ 基因密码子２４９ 特征性突变来进一步阐明该基因在ＨＣＣ 发生机制中的意义。方法：收集９ 份癌组织中经单链构型多态性分析研究证实存在密码子２４９ 突变的ＨＣＣ 标本和１１ 例无此突变的ＨＣＣ 标本,采用等位基因特异性ＰＣＲ（ＡＳ－ ＰＣＲ） 方法比较研究癌组织和癌周组织中密码子２４９ 突变情况。结果：２０ 例ＨＣＣ 中,癌周组织中密码子２４９ 特征性突变检出率为７０ ％ 。９ 例ＳＳＣＰ 阳性者的癌周组织中的特征性突变检出率达１００ ％ 。１１ 例阴性患者的癌组织和癌周组织也能检出一定水平的突变。结论：ｐ５３基因密码子２４９ 突变在非癌肝组织中广泛存在。癌组织中密码了２４９ 特征性突变可能发生在肿瘤形成之前。     

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.     

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.     

P53 mutations may be related to tumor invasiveness of human hepatocellular-carcinoma in china

A combined polymerase chain reaction (PCR) with the enzyme HaeIII restriction analysis and DNA sequencing have been employed to study the mutations at codon 249 of p53 gene in two human hepatocellular carcinoma (HCC) cell lines and 28 surgical specimens of HCC. 14 of the 28 HCC samples (50%) had p53 point mutations at codon 249. All of point mutations at codon 249 in 10 cases sequenced are AGG to AGT transversion. p53 gene mutated more frequently in invasive HCCs than that in non-invasive HCCs. This suggested that the codon 249 was a mutational hotspot of p53 gene in human HCCs in China, and p53 mutations may be related to tumor invasiveness of human HCC.     

非放射性PCR-LIS-SSCP技术快速分析肝癌患者p53基因249位密码子的突变

目的应用非放射性PCR-LIS-SSCP方法快速分析肝癌患者p53基因第249位密码子突变.方法采用PCR-LIS-SSCP方法,研究了16例来自中国广西、江苏启东和上海地区肝癌患者p53基因第249位密码子的突变情况,并用限制性内切酶和DNA测序对PCR-LIS-SSCP方法所获得的结果进行了验证.结果从16例肝癌病人中检出3例在该位点具有突变.其中广西地区的4例肝癌病人有1例发生突变,江苏启东地区的4例肝癌病人有1例发生突变,上海地区的8例肝癌病人有1例发生突变. 结论用该方法检测肝癌病人p53基因第249位密码子突变频率为18.8%(3/16),为该方法应用于候选新基因的未知突变研究奠定了基础.     

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.     

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.     

p53 gene mutation spectrum in hepatocellular carcinoma.

In order to clarify the significance of mutation of the p53 tumor suppressor gene in the genesis and development of human hepatocellular carcinoma (HCC) in an aflatoxin B1 low-exposure area, the spectrum, i.e., incidence, type, and site, of p53 gene mutations was examined in 169 tissue samples resected mainly from Japanese patients using single-strand conformation polymorphism analysis and direct sequencing. Forty-nine tumors (29%) showed a p53 mutation (39 point mutations and 10 frameshifts). The point mutations comprised 18 transitions, only 4 of which occurred at CpG sites, and 21 transversions. Two evolutionarily conserved domains, IV and V, contained 65% of all mutations and codon 249 was the most frequent mutation site (7/49). The spectrum of p53 mutation did not differ among HCCs in relation to the type of hepatitis virus infection, sex, age, and background liver disease of patients, tumor size, or presence of metastasis, but incidence and site were significantly associated with the degree of differentiation of cancer cells. In poorly differentiated HCC, p53 mutation was frequent (54%) and clustered on domains IV and V, whereas in well or moderately differentiated HCC, the mutation was less frequent (21%) and equally distributed on domains II to V. Restriction fragment length polymorphism analysis revealed loss of heterozygosity on chromosome 17p in 55 (69%) of 80 informative cases and in 34 (95%) of 36 cases with p53 mutation. Therefore, p53 gene mutation is suggested to occur independently of the type of viral infection or status of preexisting liver disease and to occur preferentially in moderately and poorly differentiated HCCs in association with or after loss of another p53 allele as a late event of HCC progression.     

p53 Abnormalities in hepatocellular carcinoma from United States patients: analysis of all 11 exons

Mutations of the tumor suppressor gene p53 have been found inhepatocellular carcinomas (HCCs) from many countries where HCCis common, but p53 mutations detected by direct sequencing inHCCs from the United States (where HCC is uncommon) have beenreported only rarely, p53 Exons 1 to 11 were analyzed by polymerasechain reaction, single-strand conformation polymorphism analysis,and direct sequencing in HCCs from 12 patients from the UnitedStates and in adjacent non-tumorous liver from 10 of them. Abnormalitiesof the p53 gene were found in five of the 12 tumors, includingmutations in exon 4 (one patient), exon 5 (one patient) andexon 8 (three patients). In all 12 tumors, a normal conformationof exon 7 was found; in five of these HCCs the absence of mutationswas confirmed by direct sequencing. Thus, the mutations of p53codon 249 that have frequently been found in HCCs from endemicareas apparently do not play a role in most HCCs in the UnitedStates. Since the mutations in codon 249 are thought to be dueto aflatoxin ingestion, the data suggest that factors otherthan aflatoxin may be responsible for the p53 abnormalitiesin the patients from the USA.     

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.     

High prevalence of mutations at codon 249 of the p53 gene in hepatocellular carcinomas from Senegal.

In hepatocellular carcinoma, mutation within the p53 gene occurs mainly at codon 249 and its frequency has been associated with exposure to aflatoxin. As Senegal is a country where liver cancer incidence is one of the highest in the world and where people are highly exposed to aflatoxin, we screened 15 liver cancer samples from this country for mutation at codon 249 of the p53 gene. Non-tumoral DNA from the patients showed a wild type genotype. Mutation at codon 249 of the p53 gene was detected in 10 of the 15 tumour tissues tested (67%). This frequency of mutation in codon 249 of the p53 gene is the highest described. These results confirmed that there is an association between countries of high aflatoxin intake and a high frequency of mutation in codon 249 of p53 gene, and that HBV alone does not contribute to these base changes.     

中国高发区肝细胞癌p53基因热点突变的规律性

目的 研究同发区肝细胞癌（ＨＣＣ）Ｐ５３基因２４９密码子高频度突变的规律性,为探寻癌及基于Ｐ５３的基因和免疫治疗提供依据。方法 用巢工ＰＣＲ结合测序和限制性片段长度多态（ＲＦＬＰ）分析的方法,研究１９９４－１９９７年收集的９７例启东和２２便北京的肝细胞癌标本中,Ｐ５３基因２４９密码子的突变率及突变的基因型。用免疫组化法研究癌及癌周细胞中Ｐ５３蛋白的表达程度。 １９９４－１９９７年,启东肝细胞癌中,     

Ser-249 p53 mutations in plasma DNA of patients with hepatocellular carcinoma from The Gambia

BACKGROUND: A selective mutation, an arginine-to-serine substitution in codon 249, of the p53 gene has been identified as a "hotspot" mutation in hepatocellular carcinoma (HCC). This mutation occurs in populations that are exposed to aflatoxins and have a high prevalence of hepatitis B virus carriers. We evaluated whether this mutation could be detected in cell-free DNA isolated from the plasma of subjects from The Gambia to detect this mutation that is strongly associated with HCC. METHODS: Fifty-three patients with HCC, 13 patients with cirrhosis, and 53 control subjects were prospectively recruited from The Gambia. Sixty patients, of non-African origin, with various liver pathologies were also selected from France. DNA was extracted and purified from 200-microL aliquots of plasma. The Ser-249 p53 mutation was detected by restriction endonuclease digestion of polymerase chain reaction products from exon 7 and was confirmed by direct sequencing of the amplified DNA. RESULTS: The Ser-249 p53 mutation was detected in plasma DNA from 19 (36%) of the 53 patients with HCC, two (15%) of the 13 patients with cirrhosis, and three (6%) of the 53 control subjects. This mutation was not detected in any plasma DNA from the European patients. The adjusted odds ratio for having the mutation was 16.4 (95% confidence interval = 3.0-90.5) for patients with HCC compared with the control subjects. CONCLUSION: The Ser-249 p53 mutation in plasma DNA is strongly associated with HCC in Gambian patients. This mutation was also detected at a much lower prevalence in plasma DNA from Gambian patients with cirrhosis and in Gambian control subjects, findings that may lead to the earlier detection of HCC. Use of the Ser-249 p53 mutation should facilitate further molecular epidemiologic studies on the development of HCC.     

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.     

Low frequency of p53 gene mutation in tumors induced by aflatoxin B1 in nonhuman primates.

Aflatoxin B1 has been suggested as a causative agent for a G to T mutation at codon 249 in the p53 gene in human hepatocellular carcinomas from southern Africa and Qidong in China. To test this hypothesis, nine tumors induced by aflatoxin B1 in nonhuman primates were analyzed for mutations in the p53 gene. These included four hepatocellular carcinomas, two cholangiocarcinomas, a spindle cell carcinoma of the bile duct, a hemangioendothelial sarcoma of the liver, and an osteogenic sarcoma of the tibia. None of the tumors showed changes at the third position of codon 249 by cleavage analysis of the HaeIII enzyme site at codon 249. A point mutation was identified in one hepatocellular carcinoma at the second position of codon 175 (G to T transversion) by sequencing analysis of the four conserved domains (II to V) in the p53 gene. These data suggest that mutations in the p53 gene are not necessary in aflatoxin B1 induced hepatocarcinogenesis in nonhuman primates. The occurrence of mutation in codon 249 of the p53 gene in selective samples of human hepatocellular cancers may indicate involvement of environmental carcinogens other than aflatoxin B1 or that hepatitis B virus-related hepatitis is a prerequisite for aflatoxin B1 induction of G to T transversion in codon 249.     

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.     

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.     

Mutations of p53 gene in hepatocellular-carcinoma (hcc) correlate with tumor progression and patient prognosis - a study of 138 patients with unifocal hcc

The mutation spectrum of p53 gene and its biological significance were studied in 138 patients with unifocal primary hepatocellular carcinoma (HCC) in Taiwan. The p53 mutations were detected in 51 cases (37%); 36 (71%) were missense mutations. The others (29%) included mutations at the intron-exon junctions (5 cases), deletion or insertion (4 cases), nonsense mutations (4 cases), and silent mutations (2 cases). The mutation sites were scattered from exons 4 to 10, predominantly (75%) in exons 5, 7, and 8. Of these mutations, 72% were transversions, mostly G:C --> T:A change (46%); while only 28% were transitions. Mutation occurred at codon 249 only in 14 cases (10%), but accounted for 27% of the mutations. The p53 mutations correlated with allele loss of p53 locus (52% vs 17% p<0.02), alpha-fetoprotein elevation (45% vs 28%, p<0.04), and poorly differentiated HCC (46% vs 10%, p<0.0001). The p53 mutation rate was two times higher in large than in small HCC (48% vs 26%, p<0.008), and in more advanced tumor (stage 3 vs stages 1 and 2: 49% vs 21%, p<0.0007). HCC patients with mutated p53 gene had a worse outcome (5-year survival; 18% vs 38%, p<0.008). We conclude that p53 gene mutation is common in advanced HCC, occurs as a late event in HCC growth, correlates with tumor progression and aggression, and is a useful molecular prognostic parameter of HCC. The p53 mutation patterns did not correlate with HBV or HCV infection. The frequency of p53 mutations did not differ between Taiwanese patients and mainland Chinese in Taiwan. However, mutation at codon 249 was more common in Taiwanese patients (p<0.05), while mutations of other types more frequent in the mainlanders (p<0.03). Hence endogenous and exogenous factors other than aflatoxin may also play a role in p53 mutation in HCC.     

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.