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Specific p53 Mutations Detected in Plasma and Tumors of Hepatocellular Carcinoma Patients by Electrospray Ionization Mass Spectrometry¹

Department of Environmental Health Sciences [P. E. J., T. W. K., J. D. G.]; Department of Oncology [T. W. K., B. V., J. D. G.], Johns Hopkins Medical Institutions, Baltimore, MD 21205; Shanghai Cancer Institute, Shanghai, 200032 People’s Republic of China [G-S. Q., Y. W.]; International Agency for Research on Cancer, Lyon 69372, France [M. D. F.]; Qidong Liver Cancer Institute, Qidong, Jiangsu Province, 226200 People’s Republic of China [Y-R. Z, P. L., J-B. W.]; Howard Hughes Medical Institute, Baltimore, MD 21205 [B. V.]

	ABSTRACT

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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.

	Introduction

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HCC³ is one of the most common cancers worldwide, and there is a striking geographical variation in incidence. In the People’s Republic of China, HCC accounts for over 250,000 deaths annually with an incidence rate in some areas of the country approaching 150 cases/100,000/year. Etiological factors that have been associated with the development of the disease include infection with the hepatitis B virus or hepatitis C virus and exposure to high levels of dietary AFB₁ (1 , 2) .

Mutations in the p53 tumor-suppressor gene have been identified in a majority of human cancers, and distinct mutational spectra are observed within this gene across cancers of different tissues (3) . The most striking example of a specific mutation in the p53 gene is a GT transversion in the third base of codon 249, which has been detected in 10–70% of HCCs from areas with a high exposure to AFB₁, whereas this mutation is absent from HCC in regions with negligible exposure to AFB₁ (4, 5, 6) . Studies in bacteria have shown that aflatoxin exposure causes almost exclusively GT transversions, providing support for the implication that aflatoxin causes this specific mutation (7) . Furthermore, it has been shown that the aflatoxin-epoxide can bind to codon 249 of p53 in vitro (8) , and human hepatocarcinoma cells exposed to aflatoxin in the presence of rat liver microsomes had a high prevalence of GT transversions in codon 249 of the p53 gene (9 , 10) .

Recently, we have developed a sensitive and specific method for detection of defined genetic variants in PCR-amplified products of the APC gene using ESI-MS (11) . In this paper, we report on the application of SOMA to the analysis of p53 mutations in tumor DNA from HCC patients from Qidong and Shanghai and provide a comparison of SOMA to DNA sequencing. Additionally, tumor and plasma pairs from 20 cases of HCC have been analyzed and show for the first time a relationship between the occurrence of this mutation in tumor tissue and its presence in blood circulation.

	Materials and Methods

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Case Materials.
Liver tumor and plasma samples were obtained as part of an ongoing prospective cohort investigation of liver cancer in high-risk areas of China. This collaboration between the Shanghai Cancer Institute, the Qidong Liver Cancer Institute, and Johns Hopkins University has been approved by each of the respective Institutional Review Boards for Human Research. The p53 mutational analysis by DNA sequencing in the initial 25 tumor samples has been reported previously (12) . Healthy normal plasma samples were collected from individuals in the United States in an independent study for the analysis of dietary exposures, and this was also approved by the Johns Hopkins University Institutional Review Boards for Human Research.

DNA Extraction.
Genomic DNA was prepared from frozen liver tissue using a QIAamp Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer’s recommendations.

Blood samples were collected in EDTA-containing tubes and plasma was transferred to a plain tube and stored at -70°C until further processing. DNA was extracted from plasma using a QIAamp Blood Kit (Qiagen) according to the manufacturer’s protocol. A final elution volume of 50 µl was used. DNA was isolated from 100 µl of plasma for the Chinese samples and from 300 µl of plasma for the normal controls.

PCR.
Primers used for PCR amplification were as follows: (a) p53–8F1: 5'-CTACAACTACATGTGTAACAGCTGGAGCATGGGCGGCATGAAC-3'; and (b) p53–8R1: 5'-CTGGAGTCTTCCACTGGAGTGATGGTGAGGATG-3'.

The expected size of the product was 84 bp. PCR was performed as described previously (13) . Reactions were performed with 25–50 ng of genomic DNA from liver tissue or with 10 µl of DNA eluate from plasma samples. The final reaction volume was 50 µl, and thermocycling conditions were 95°C for 2 min and then 40 cycles of 95°C for 30 s, 65°C for 30 s, and 72°C for 30 s.

Preparation and Purification of Short Oligonucleotides.
Digestion of the PCR products with BpmI restriction enzyme and subsequent purification of the fragments were performed as described previously (11) . The size of the internal fragments after BpmI digestion was 8 bp. These fragments were then subjected to HPLC as described previously (11) , with the modification that the column used was a 1 x 150 mm YMC ODS-AQ C18 reversed phase column (5 µM, 120Å pore size; Waters Corp., Milford, MA), and the gradient used was from 60% A:40% B programmed to 40% A:60% B in 5 min. Solvent A was 0.4 M 1,1,1,3,3,3-hexafluoro-2-propanol (pH6.9), and solvent B was 50:50 (v:v) 0.8 M 1,1,1,3,3,3-hexafluoro-2-propanol:methanol.

ESI-MS.
Mass spectra were obtained with a LCQ ion-trap mass spectrometer (Thermoquest; Finnigan MAT, San Jose, CA) equipped with an ESI source operated in the negative ionization mode. The instrument was tuned daily by infusion at 3 µl/min of a synthetic oligonucleotide (10 ng/µl in 60% A:40% B) into the HPLC mobile phase through a low dead-volume tee. Typical settings for the spray voltage were -3.0 to -4.0 kV, and the heated capillary was held at 150°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 m/z 750 to m/z 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 msec; isolation width, 3 Da) using six scan events monitoring each [M-2H]^2- oligonucleotide individually [Scan event 1, AGG-s (5'-CGGAGGCC-3'), m/z 1256.3750–2000; scan event 2, AGG-as (5'-CCTCCGGT-3'), m/z 1219.8750–2000; scan event 3, AGT-s (5'-CGGAGTCC-3'), m/z 1244.3750–2000; scan event 4, AGT-as (5'-ACTCCGGT-3'), m/z 1231.8750–2000; scan event 5, AGA-s (5'-CGGAGACC-3'), m/z 1248.8750–2000; and scan event 6, AGA-as (5'-TCTCCGGT-3'), m/z 1227.3750–2000]. Analysis of tumor/plasma pairs was performed using only the first four scan events. Reconstructed ion chromatograms were generated and smoothed from this raw data using an isolation width of 1.0 Da and normalized to the largest of the six oligonucleotide ion peaks. The fragment ions used for each oligonucleotide were: AGG-s, m/z 1047.3 + 1180.7 + 1566.0; AGG-as, m/z 1268.6 + 1347.8 + 1637.2; AGT-s, m/z 899.2 + 1437.4 + 1542.4; AGT-as, m/z 1075; AGA-s, m/z 1404.0 + 1693.1; and AGA-as, m/z 979.3 + 1286.0 + 1652.2. A sample was considered positive for a mutation if there was a signal in both the mutant sense and mutant antisense channels and if the signal was >3% when normalized to the AGG-as peak.

	Results

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Comparison of SOMA and DNA Sequencing for the Detection of p53 Mutations.
Initial experiments were performed to develop a strategy for an ESI-MS/MS analysis to measure AGGAGT and AGGAGA mutations in codon 249 of the p53 gene. For this, SOMA was used in a masked study to analyze 25 HCC samples from patients in Shanghai and Qidong that had been analyzed previously by more traditional methods. The DNA samples were sequenced for mutations in the p53 gene, and 10 of 24 (41.7%) had a detectable GT mutation in codon 249 with a readable sequence not generated for one sample (12) . For the SOMA technique, PCR primers were designed to generate 8-mer oligonucleotides after PCR amplification and BpmI restriction digestion. These fragments were then suitable for analysis using HPLC-ESI-MS/MS. Typical chromatograms for samples with and without a mutation are shown in Fig. 1, A and B . Samples containing only the wild-type sequence had a signal in the channels monitoring masses representative of both the wild-type (AGG) sense and antisense alleles (Fig. 1A) . For samples with a GT mutation at the third base of codon 249, peaks were detected in the sense and antisense channels for both the wild-type (AGG) alleles and the GT mutant alleles (AGT) (Fig. 1B) .

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. ESI-MS/MS analysis of DNA from two separate HCCs for mutations in codon 249 of the p53 gene. The six mass chromatograms for each patient represent the sense (s) and antisense (as) masses for the wild-type sequence (AGG), the AGT mutation, and the AGA mutation. A, patient sample with wild-type alleles only. B, patient sample with a GT mutation.

p53 Mutations in Liver Tumors and Paired Plasma Samples.
A prospective cohort investigation is being conducted in Qidong, People’s Republic of China, to examine risk factors for the development of liver cancer. In this study, 2200 men and women have been followed for 8 years and plasma samples collected every 12 months. Twenty liver tumor and plasma pairs obtained at the time of cancer diagnosis arising within this cohort were analyzed for GT mutations in codon 249 of the p53. Similar to the prevalence of p53 mutations reported in our previous study (12) , 11 of 20 tumor samples contained an AGGAGT mutation in codon 249 of the p53 gene. Each sample was adjudged to be positive for the p53 mutation when a signal in both the sense and antisense AGT channel was recorded. Paired plasma samples were also analyzed, and 6 of 11 (55%) patients with a codon 249 mutation in the tumor tissue had a positive plasma sample for the mutation. Representative data are shown in Fig. 2 . Five of the nine patients lacking the p53 mutation in their tumor also had no detectable levels of this DNA in their plasma; however, four plasma samples had detectable levels of the codon 249 mutation, whereas none was detected in the liver tissue, suggesting a possible second occult HCC. Ten plasma samples from healthy, normal individuals from the United States were also analyzed, and all of these samples were negative for the p53 specific mutation. In this sample set, a 3-fold larger sample of plasma (300 µl) was used for DNA isolation to achieve a robust wild-type signal. This analysis establishes for the first time a direct link between p53 mutations in liver tumors and detection in plasma samples.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. ESI-MS/MS analysis of DNA from tumor and plasma pairs from two patients with HCC for the presence of the GT mutation at codon 249 of the p53 gene. The four mass chromatograms for each sample represent the sense (s) and antisense (as) masses for the wild-type (AGG) sequence and the AGT mutation. Patient I has a mutation in both tumor and plasma DNA, whereas patient II does not have a detectable mutation in either tumor or plasma DNA.

	Discussion

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A higher percentage of the tumors from Qidong had a GT mutation at the third base of codon 249 of the p53 gene than tumors from Shanghai (46.7% compared with 30%). The mutation frequency corresponds to exposure to aflatoxins because these areas have high and intermediate exposure levels, respectively. The frequency of mutations in the samples from Qidong is similar to that found in other studies (5 , 14) . However, for HCC cases from Shanghai, lower mutation frequencies have been reported (14) .

AFB₁ has been observed to induce GT and GA mutations at approximately the same frequency in a plasmid-based system with a lacZ' mutational target (15 , 16) , and GA mutations have also been detected at the third base of codon 249 of the p53 gene in HepG2 cells exposed to aflatoxin in the presence of rat liver microsomes (9) . It was therefore of interest to determine whether GA mutations were present in tumor or normal tissue from HCC patients using SOMA. This could easily be achieved by monitoring masses representing alleles with a GA transition during the same injection, and therefore did not require additional sample analysis time. No GA mutations were detected in any of the normal or tumor samples, indicating this mutation is not common in human HCC cases. There have been no previous reports that this mutation has been found in human HCC cases. Because the substitution of a GA at the third base of codon 249 does not change the amino acid in the protein, this mutation is, therefore, unlikely to provide a selective advantage even if it were formed and subsequent samples were not analyzed for this mutation.

Whereas the detection of specific p53 mutations in liver tumors has provided insight into the etiology of certain liver cancers, the application of these specific mutations to the early detection of cancer offers great promise for prevention (17) . A recent report by Kirk et al. (18) reported for the first time the detection of codon 249 p53 mutations in the plasma of liver tumor patients from the Gambia; however, the mutational status of the tumors was not known. These authors also reported a small number of cirrhosis patients having this mutation, and given the strong relationship between cirrhosis and the future development of HCC, the possibility of this mutation being an early detection marker needs to be explored. In this study, we have reported for the first time the relation of plasma and tumor pairs for the occurrence of specific p53 mutations. The presence of a detectable mutation in the plasma when the tumor tissue had only wild-type alleles could be indicative of multiple independent HCC nodules in these patients. The absence of a mutation in the plasma sample from a patient whose tumor was found to have a mutation may be attributable to insufficient sensitivity. Thus, SOMA is a highly accurate method for detection of specific mutations in human tumor samples, and future prospective studies will determine the practical limit of detection. The signal that is measured is quantitative in nature, and we therefore envisage future applications of SOMA in the quantitation of mutations in samples containing low levels of mutant allele in a background of normal alleles. In future studies, the predictive power of this marker to assess HCC development will be investigated.

	ACKNOWLEDGMENTS

 
We thank Drs. Asif Rashid for the sequencing data and Paul Strickland for his contributions to this investigation.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Financial support for this work was provided by the Clayton Fund, Grant P01 ES06052; National Institute of Environmental Health Sciences Center P30 ES03819; National Cancer Institute Center P30 CA06973; National Cancer Institute Grants CA43460, CA57345, and CA 62924; and Association for International Cancer Research Grant 94275.

² To whom requests for reprints should be addressed, at Department of Environmental Health Sciences, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205

³ The abbreviations used are: HCC, hepatocellular carcinoma; SOMA, short oligonucleotide mass analysis; ESI-MS, electrospray ionization mass spectrometry; ESI-MS/MS, electrospray ionization tandem mass spectrometry; AFB₁, aflatoxin B₁; HPLC, high-pressure liquid chromatography.

Received 8/16/00. Accepted 11/13/00.

	REFERENCES

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jackson, P. E. Articles by Groopman, J. D. Search for Related Content PubMed PubMed Citation Articles by Jackson, P. E. Articles by Groopman, J. D.