Hepatitis B 1762^T/1764^A Mutations, Hepatitis C Infection, and Codon 249 p53 Mutations in Hepatocellular Carcinomas from Thailand

Hepatocellular carcinoma (HCC) is a major cause of cancer morbidity and mortality in many parts of the world, including Asia and sub-Saharan Africa, where there are >600,000 new cases each year (1, 2). Cancer registry data show that HCC is the leading cause of cancer mortality in Thailand (3). The major known etiologic factors associated with development of HCC in these regions are infection with hepatitis B (HBV) and/or hepatitis C (HCV) virus and lifetime exposure to high levels of aflatoxin B₁ in the diet (4-6). Detailed knowledge of the etiology of HCC has spurred many mechanistic studies to understand the pathogenesis of this nearly always fatal disease, and this knowledge is beginning to be translated to preventive interventions in high-risk populations.

HBV is a significant risk factor for HCC in the developing world where there are >400 million viral carriers (7, 8). The biology, mode of transmission, and epidemiology of this virus continues to be actively investigated and has been recently reviewed (7, 8). The contribution of HBV to the pathogenesis of liver cancer is multifactorial and is complicated by the identification of mutant variants of HBV that modulate the carcinogenesis process (4, 9, 10). The HBV genome encodes its essential genes with overlapping open reading frames; therefore, a mutation in the HBV genome can alter the expression of multiple proteins. In many cases of HCC in China and Africa, a double mutation in the HBV genome, an adenine to thymine transversion at nucleotide 1762, and a guanine to adenine transition at nucleotide 1764 (1762^T/1764^A) has been found in tumors (7, 11, 12). The onset of these mutations has also been associated with increasing severity of the HBV infection and cirrhosis (11, 12). Thus, the tracking of this mutation with disease outcomes makes it a candidate biomarker for the early detection of HCC risk in individuals. Finally, the emergence of HCV infection as an etiologic factor in HCC raises the potential for further viral-viral and viral-chemical interactions in the pathogenesis of this disease (6, 13).

Several studies have now shown that DNA isolated from serum and plasma of patients with cancer contains the same genetic aberrations as DNA isolated from an individual's tumor (14-17). The process by which tumor DNA is released into circulating blood is unclear but may result from accelerated necrosis, apoptosis, or other processes (18). Recently, we have found that a specific codon 249 p53 mutation and an HBV 1762^T/1764^A double mutation were not only detectable in plasma samples at the time of HCC diagnosis, but that they could be measured in some individuals at least 8 years before diagnosis (19, 20). To better understand the interactions and contributions of these viral and chemical agents to the risk of HCC, their distribution must be evaluated in several populations who are at risk of HCC. In this study we have used mass spectrometry to measure the occurrence of an HBV 1762^T/1764^A double mutation and an aflatoxin-specific mutation of the p53 gene, a guanine to thymine transversion at codon 249; in addition, biomarkers of HCV infection were also measured in HCC cases and controls from Chiang Mai, Thailand.

All HCC cases were diagnosed at Chiang Mai University using serology, ultrasound, and/or histopathology. The age and gender characteristics of the 25 HCC cases and the 102 subjects in the ongoing case-control study are found in Tables 1 and 2, respectively. The overrepresentation of male cases is consistent with previous distributions of HCC in Asia (6). A case-control study consisting of 34 HCC cases were age and gender matched with 68 hospital-based and liver disease–free control subjects. Other characteristics of the cases and controls are shown in Table 2. Plasma samples were coded before analysis. The serology studies to ascertain hepatitis B surface antigen and HCV status was done using commercially available assays. This research has been reviewed and approved by the institutional review boards at Johns Hopkins University and Chiang Mai University.

DNA was isolated from plasma samples using Qiagen columns (Valencia, CA) as previously described (17, 20). DNA was also extracted from paraffin-embedded tissue specimens after sectioning and isolation using the Pinpoint Slide DNA Isolation System (Zymo Research, Orange CA).

Short oligonucleotide mass analysis for HBV and p53 mutations involved a PCR enrichment step. The primers for the HBV mutation analysis were HBVx-7F, 5′-TTT GTT TAA AGA CTG GGA GGA CTG GAG GGA GGA GAT TAG GTT A-3′, and HBVx7R, 5′-TGG TGC GCA GAC CAA TTT ATG CTG GAG GCC TCC TAG TAC AA-3′. The PCR primers for the p53 analysis were p53-8F1, 5′-CTACAACTACATGTGTAACAGCTGGAGCATGGGCGGCATGAAC-3′, and p53-8R1, 5′-CTGGAGTCTTCCACTGGAGTGATGGTGAGGATG-3′. In both cases the thermocycling conditions were 95°C for 2 minutes, then 40 cycles of 94°C for 30 seconds, 65°C for 30 seconds, and 72°C for 30 seconds followed by a final extension of 72°C for 2 minutes. Negative controls (no DNA added) were included for each set of PCR reactions. PCR product was purified by ethanol precipitation and digested with 8 units of BpmI (New England Biolabs, Beverly, MA) overnight at 37°C in a volume of 50 μL to release internal fragments. These fragments were 7 bp for HBV and 8 bp for p53. A phenol-chloroform extraction followed by an ethanol precipitation in the presence of SeeDNA (Amersham Pharmacia, Piscataway, NJ) was done to purify samples for analysis by electrospray ionization-mass spectrometry.

The digested fragments were resuspended in 10 μL of the high-performance liquid chromatography (HPLC) mobile phase [70:30 (v/v) solvent A/solvent B, in which solvent A was 0.4 mol/L 1,1,1,3,3,3-hexofluoro-2-propanol (pH 6.9) and solvent B was 50:50 (v/v) 0.8 mol/L 1,1,1,3,3,3-hexafluoro-2-propanol:methanol] and 8 μL was introduced into the HPLC coupled to the electrospray-mass spectrometry. HPLC was carried out at 30 μL/min using a 1 × 150-mm Luna C18, 5 μ reversed phase column (Phenomenex, Torrance, CA) and Surveyor pumps (ThermoFinnigan Corp, San Jose, CA). The gradient conditions were 70% A/30% B isocratic 1 minute programmed to 100% B in 3 minutes, in which it was held for 2.5 minutes followed by a return to 70% A/30% B in 1.5 minutes and isocratic elution for the remaining 32 minutes of the chromatography.

Mass spectra were obtained with a LCQ Deca ion-trap mass spectrometer (ThermoFinnigan Corp) equipped with an electrospray ionization source operated in the negative ionization mode. The spray voltage was set at −4.0 kV and the heated capillary was held at 240°C. Each of the oligonucleotide ions was isolated in turn and subjected to collision-induced dissociation at 30% collision energy. Full scan spectra of the resultant fragment ions from relative intensity (m/z) 600 to 2000 were acquired, and signals from up to three specific fragment ions were summed as a function of time for each of the oligonucleotides.

The mass spectrometer was programmed to acquire data in the centroid mode (1 μscan, 200 milliseconds, isolation width 3 Da) using scan events monitoring each [M-2H]²⁻ oligonucleotide individually [scan event 1: WT-s (5′-AAGGTCT-3′), m/z 1099.20→750-2000; scan event 2: WT-as (5′-ACCTTTA-3′), m/z 1066.70→750-2000; scan event 3: Mut-s (5′-ATGATCT-3′), m/z 1086.70→750-2000; scan event 4: Mut-as (5′-ATCATTA-3′), m/z 1078.70→750-2000]. The fragment ions used for each oligonucleotide were WT-s (m/z 803.78 + 1132.22 + 1243.27), WT-as (m/z 910.07 + 1531.29), Mut-s (m/z 914.30 + 1227.29), and Mut-as (m/z 1084.0+1252.0). A sample was considered positive when fragments were observed in either or both sense and antisense channels for the mutant allele in at least three scans across the peak.

The mass spectrometer was programmed to acquire data in the centroid mode (1 μscan, 200 milliseconds, isolation width 3 Da) using four scan events monitoring each [M-2H]²⁻ oligonucleotide individually [scan event 1: AGG-s (5′-CGGAGCCC-3′), m/z 1256.3→600-2000; scan event 2: AGG-as (5′-CCTCCGGT-3′), m/z 1219.8→600-2000; scan event 3: AGT-s (5′-CGGAGTCC-3′), m/z 1244.3→600-2000; scan event 4: AGT-as (5′-ACTCCGGT-3′), m/z 1231.8→600-2000]. Reconstructed ion chromatograms were generated and smoothed from this raw data using an isolation width of 1.0 Da. The fragment ions used for each oligonucleotide were AGG-s (m/z 1047.3 + 1180.7), AGG-as (m/z 1268.6 + 1347.8 + 1637.2), AGT-s (m/z 1437.4 + 1542.4), and AGT-as (m/z 1075.0). A sample was considered positive when fragments were observed in either or both sense and antisense channels for the mutant allele in at least three scans across the peak.

Serologic evidence of HBV infection was documented using the licensed enzyme immunoassays for hepatitis B surface antigen, hepatitis B core antigen, and anti–hepatitis B core (Abbott Laboratories, Abbott Park, IL). Antibodies to HCV were measured using a third-generation licensed HCV enzyme immunoassay (EIA, Abbott Laboratories); HCV RNA was amplified using HCV core E1 primers according to methods previously reported (21, 22).

All plasma samples were coded to mask the case status for short oligonucleotide mass analysis and interpretation. Standard descriptive analyses were conducted. Frequency distributions and proportions were calculated for categorical variables; medians and SDs were calculated for continuous variables. Cases and controls were compared using simple conditional logistic regression to account for the matched design.

Initial studies in this investigation involved the assessment of the status of both HBV and p53 mutations in 25 liver tumors. Nineteen (76%) of 25 tumors contained detectable integrated HBV. Of these 19 HBV positive tumors, 17 (89.5%) had an HBV 1762^T/1764^A double mutation. Tumor and adjacent normal liver tissue pairs were available for four of these cases and, in each instance, the HBV mutation was present in both samples. The median age of the HCC cases in this group was 54.2 years; the SD was 13.5 years (Table 1).

In contrast to the HBV mutation data, the codon 249 p53 guanine to thymine transversion mutation was detected in 6 (24%) of 25 tumors. In the four tumor/normal pairs, the p53 mutation was detected in 3 of the tumors but not in any of the adjacent normal tissues. Furthermore, the median age of the liver cancer cases containing the codon 249 p53 mutation was 50.1 years, which was nearly 4 years younger than the whole group. Both the HBV and p53 mutations were detected in 4 (16%) of the samples. HCV virus status was measured in 20 of the tumor samples from paired plasma specimens and 4 (20%) of the 20 were positive for HCV antibody; in each of these cases the tumor contained the HBV 1762^T/1764^A double mutation. The high prevalence of all of these markers of HCC risk encouraged the design of a case-control investigation.

Plasma samples from 14 (41.2%) of the 34 HCC cases were found to have detectable levels of HBV DNA. All 14 of the plasma HCC samples were positive for the HBV 1762^T/1764^A mutation. Five (7.4%) of the 68 control plasma samples had detectable levels of HBV DNA; 2 (2.9%) were positive for the HBV double mutation. Among HBV DNA positive samples, a statistically significant (P = 0.01, two-tailed) overrepresentation of the HBV double mutation was observed among cases. Nine (26.5%) of the 34 cases and 10 (14.7%) of the 68 controls were positive for the codon 249 p53 mutation. Five cases (14.7%) and no controls were positive for both mutations. Six (17.7%) of the cases and 1 (1.5%) of the controls were EIA positive for HCV. Three cases (8.8%) and 1 control (1.5%) were HCV-RNA positive. Although the occurrence of the HBV 1762^T/1764^A mutation had been previously reported in patients with chronic liver disease in Thailand, this case-control study extends these findings to HCC patients. Five of the HCC cases contained both the HBV 1762^T/1764^A and codon 249 p53 mutations in plasma and the median age of diagnosis of cancer in these patients was 34.1 years compared with 51.5 years for the other HCC cases. A liver cancer case comparison analysis was done as shown in Table 3. At the time of the liver cancer diagnosis, 41.2% and 26.5% of the cases were positive for the HBV 1762^T/1764^A and codon 249 p53 mutations in plasma, respectively. Of the 16 cases containing neither the HBV or p53 mutation, 3 (18.8%) were EIA positive for HCV biomarker. In addition, none of the cases had all three markers and only one of the cases showed the presence of the HBV mutation and coinfection with HCV. None of the controls had both mutations. Among the 13 HCC cases who were negative for HBV DNA, HCV, and a p53 mutation, 11 were hepatitis B surface antigen seropositive, 9 had a >20-year history of tobacco smoking, 6 had used alcohol heavily for >20 years, 2 used betel nut, and 2 were type 2 diabetics.

Major risk factors for HCC include chronic infection with HBV or HCV and dietary exposure to aflatoxin B₁, a potent, naturally occurring liver carcinogen (6, 23). Because the etiology of HCC is complex, the disease commonly progresses through a multistage process, with most cases involving liver cirrhosis (23). Studies in some populations in Asia and Africa suggest that a specific double mutation in the HBV X gene may be associated with accelerated carcinogenesis among HBV carriers (2). However, the prevalence of this mutation has not been evaluated in HCC cases in Thailand. The worldwide geographic distribution of HCC illustrates that populations in which viral hepatitis infection due to HBV or HCV is endemic also have a high incidence of disease. HBV has a higher prevalence worldwide at this time and there are >400 million HBV carriers worldwide (8). Because there is a <20% chronicity rate for HBV infection, >2 billion people have been infected with this virus at some point during their lifetime. In hyperendemic areas, such as Korea and China, infection rates of HBV have exceeded 50% by age 30 and perinatal transmission accounts for 35% to 50% of HBV carriers (24, 25). HCV seems to be associated with HCC in regions with relatively low prevalence of HBV infection. In China, HCV is found in <5% of HCC cases (26). Unfortunately, HCV infection results in much higher incidence of chronicity and cirrhosis and is beginning to contribute to the major increase in HCC in countries such as Japan and the United States (2, 4). Cancer registry data show that HCC is the leading cause of cancer mortality in Thailand (3).

The etiology of HCC in some of the highest risk regions is further complicated by the strong mutiplicative interaction between aflatoxin B₁ exposure and HBV. Two cohort studies have showed this chemical-viral impact for the development of HCC. The first report resulted from monitoring and follow-up of a cohort of over 18,000 people in Shanghai (5, 27). A nested case-control study within this cohort revealed statistically significant increases in HCC among persons who had either aflatoxin exposure or HBV infection alone. For those people who had both aflatoxin and HBV exposure there was a multiplicative interaction resulting in a relative risk for developing HCC of ∼60. A subsequent nested case-control study from a cohort of >15,000 people in Taiwan also found a strong interaction between aflatoxin and HBV infection (28). Data from our study population also suggest that HBV and aflatoxin exposures may interact to promote HCC carcinogenesis, because HCC cases who had both a HBV mutation and a p53 mutation signaling aflatoxin exposure were 20 years younger when HCC was diagnosed. These findings have encouraged the development and validation of both aflatoxin and HBV biomarkers that can be used to identify high-risk individuals before HCC diagnosis.

In this study we have used mass spectrometry to measure the occurrence of an HBV 1762^T/1764^A double mutation and an aflatoxin-specific mutation of the p53 gene, a guanine to thymine transversion at codon 249; in addition, chronic infection with HCV was also measured in these cases and controls from Chiang Mai, Thailand. Induced mutations in both the integrated HBV virus genome and the p53 tumor suppressor gene have been frequently detected in liver tumors of patients from both Asia and sub-Saharan Africa (6, 7). The prevalence of these acquired mutations varies across populations and this is potentially driven in part by subtle etiologic differences in the pathobiology of hepatocellular carcinoma. Initial studies in this investigation involved the assessment of the status of both HBV and p53 mutations in liver tumors. Nearly 90% of the tumors had an HBV 1762^T/1764^A double mutation; these findings are consistent with previous results from Qidong, People's Republic of China (20). In contrast to the HBV mutation data, the codon 249 p53 guanine to thymine transversion mutation was detected in about 25% of the tumors. The level of the p53 mutations is somewhat higher than a previous report in Thailand (29). HCV virus status was measured in 19 of the tumor samples from paired plasma specimens. Only 21% were positive for HCV antibody and, in each instance, the tumor contained the HBV 1762^T/1764^A double mutation. The high prevalence of all of these markers of HCC risk encouraged us to design a case-control study.

The pathobiology of HBV infection is modulated through the selection and expression of several common viral mutants that affect a number of key viral proteins (9, 10, 30, 31). One of these common mutations is HBV 1762^T/1764^A that affects the expression of both the hepatitis B e antigen, because the mutation lies in the basic core promoter, and the X gene (32). This double mutation induces an increased inflammatory response that becomes stronger as the liver damage progresses from chronic hepatitis to cirrhosis (33). The underlying mechanism of the effects of HBV e antigen on the biology of inflammation and cirrhosis is still unclear, but substantial data suggest immune tolerance in the presence of this protein (32-34). The HBV 1762^T/1764^A double mutation also affects the amino acid sequence of the HBV X gene because it resides in codons 130 and 131, thereby inducing lysine to methionine and valine to isoleucine alterations, respectively (35). The X gene protein has been found to have numerous biological activities, but its specific role and that of this mutant protein in the pathogenesis of liver cancer have yet to be elucidated (36). The 1762^T/1764^A double mutation has been reported to occur more frequently in people infected with the genotype C strains of HBV, which is the most common genotype found in East Asian patients (37-39).

Chronic infections with either HBV or HCV have been reported to be associated with an increased risk of HCC. HBV is not cytotoxic for infected hepatocytes and these cells synthesize and secrete high levels of the S gene product (hepatitis B surface antigen), which appears in blood before the onset of symptoms. Plasma hepatitis B surface antigen levels peak during the symptomatic phase and then decline to undetectable levels within 6 months after infection in persons who clear the acute infection. HBV carrier status is then defined by hepatitis B surface antigen positivity in sequential samples obtained 6 months apart (8). Hepatitis B e antigen, HBV-DNA, and DNA polymerase also appear in the serum soon after hepatitis B surface antigen and these are all biomarkers of active viral replication (40). Thus, these plasma HBV specific biomarkers reflect an intrinsic risk for the future development of HCC, but <10% of all chronic carriers of HBV will develop this cancer. In contrast, HCV has a much higher chronicity rate and ∼80% of all infected individuals become carriers (4, 13). It is predicted that between 20% and 25% of all HCV carriers who develop cirrhosis will develop HCC subsequently.

The use of a biomarker in blood for the early detection of HCC is well established using α-fetoprotein (41). Whereas the use of α-fetoprotein as a HCC diagnostic marker is widely used in high-risk areas because of its ease of use and low cost, this marker suffers from both low specificity and sensitivity (42, 43). This lack of specificity and sensitivity has contributed to the identification of other molecular biomarkers that are possibly more mechanistically associated with HCC development. Jackson et al. (19) have showed the potential use of specific p53 mutations in blood as a biomarker of HCC risk. In a recent investigation, the temporality of detecting the HBV 1762^T/1764^A mutation and wild-type HBV in plasma and tumor specimens, both before and after the clinical diagnosis of HCC was examined (20). Similar to previous reports, almost all of the HCC tumors contained either the HBV tandem mutation or integrated HBV DNA; the HBV 1762^T/1764^A double mutation in plasma was a predictive biomarker for HCC development.

Previously, in patients in Thailand with chronic liver disease the HBV 1762^T/1764^A mutation has been examined in plasma from hepatitis B e antigen positive and negative individuals (44). These double mutations at positions 1762 and 1764 of the core promoter were found in 25 (69.4%) of 36 and 19 (76%) of 25 of the hepatitis B e antigen positive and negative individuals, respectively. Follow-up analysis of an additional 80 cases of chronic liver disease confirmed the high prevalence of this mutation in hepatitis B e antigen negative individuals (45). The study reported in this investigation extends these findings for the first time to HCC cases in Thailand.

At the time of the liver cancer diagnosis, 38% and 20.6% of the cases were positive for the HBV 1762^T/1764^A and codon 249 p53 mutations in plasma, respectively. In contrast, in Qidong, People's Republic of China, a similar case study found 73.3% and 60% of the samples had the HBV 1762^T/1764^A and codon 249 p53 mutations in plasma, respectively (17, 20). This suggest the occurrence of similar etiologic factors but at a lower prevalence in the Thailand population compared with the liver patients with cancer in Qidong, China. In contrast, the prevalence of markers of HCV infection were more frequent among HCC cases in Thailand. Because both changes are induced mutations that contribute in some fraction to the pathogenesis of HCC, these biomarkers could be useful for the early detection of an increased risk of HCC. Thus, specific biomarkers of HBV, HCV, and aflatoxin-induced changes in p53 reveal the complexity of the risk factors that contribute to hepatocellular carcinoma in northern Thailand and suggest the further evaluation of these biomarkers.