1 Department of Health Research and Policy,
2 Department of Medicine,
3 Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA,
4 Department of Pathology and
5 Division of Research, Kaiser Permanente Medical Care Program, Oakland, CA 94611, USA,
6 Metabolism Branch, National Cancer Institute, Bethesda, MD 20892, USA and
7 Orentreich Foundation for the Advancement of Science, Inc., Cold Spring-on-Hudson, NY 10516, USA
Abstract
Infection with Helicobacter pylori (H. pylori) increases stomach cancer risk. Helicobacter pylori strains with the cag pathogenicity island (PAI) induce more severe inflammation in the gastric epithelium and are more strongly associated with stomach cancer risk than strains lacking the PAI. We examined whether the prevalence of somatic p53 mutation in gastric adenocarcinoma differed between subjects with and without infection with CagA+ (a marker for the PAI) H. pylori strains. DNA from 105 microdissected tumor specimens was analyzed for mutation in exons 58 of the p53 gene by polymerase chain reaction-based single-strand conformation polymorphism followed by direct DNA sequencing. Enzyme-linked immunosorbent assays for IgG antibodies against H. pylori and CagA were performed on sera collected 231 years prior to cancer diagnosis. Tumors from CagA+ subjects were significantly more likely to have p53 mutations than tumors from CagA- subjects (including H. pylori and H. pylori+/CagA-): odds ratio = 3.72; 95% confidence interval, 1.0613.07 after adjustment for histologic type and anatomic subsite of tumor and age at diagnosis and sex of subjects. Mutations were predominantly insertions and deletions (43%) as well as transition mutations at CpG dinucleotides (33%). The data suggest that CagA+ H. pylori infection, when compared with CagA- infection or the absence of H. pylori infection, is associated with a higher prevalence of p53 mutation in gastric adenocarcinoma.
Abbreviations: CI, confidence interval; ELISA, enzyme-linked immunosorbent assay; GEJ, gastroesophageal junction; H&E, hematoxylin and eosin; ICD-9, The Ninth Revision of the International Classification of Diseases; IHC, immunohistochemistry; KPMCP, Kaiser Permanente Medical Care Program; MHC, multi-phasic health check up; OR, odds ratio; PAI, pathogenicity island; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; SSCP, single-strand conformation polymorphism.
Introduction
Stomach cancer is the second most frequent cancer in the world (1). Although age-standardized mortality rates have declined steadily over the last few decades even in high-risk countries (2), stomach cancer still accounts for a large proportion of cancer cases among both men and women in Asia, Latin America and some countries in Europe (1). Descriptive epidemiology of gastric adenocarcinoma, the most common form of stomach cancer, shows notable time trends with regard to histologic type and anatomic subsite. Laurén (3) classified gastric adenocarcinoma into intestinal and diffuse types according to histopathological features of tumor. The decrease in intestinal-type tumors largely accounts for the downward time trends of stomach cancer incidence (4), suggesting that carcinogenesis of intestinal-type tumors is more strongly affected by exogenous risk factors than that of diffuse-type tumors (5). As for the anatomic subsite, incidence of cancer in the gastroesophageal junction (GEJ) and gastric cardia has increased in the US while incidence of distal stomach cancer has decreased (6).
Infection with Helicobacter pylori (H. pylori) has been established as a risk factor for gastric adenocarcinoma (7). Helicobacter pylori strains with the cag pathogenicity island (PAI) induce particularly intense inflammatory responses of the gastric epithelium (8,9). This inflammation, in turn, is a hypothesized mechanism by which H. pylori infection leads to atrophic gastritis and gastric cancer (2,10). Helicobacter pylori strains with the cag PAI produce an immunogenic high molecular weight protein, cytotoxin-associated gene product A (CagA), and infection with such strains can be detected by a serological test for anti-CagA antibodies (11). Recently, four studies (1215) demonstrated that CagA entered gastric epithelial cells and, after phosphorylation, triggered changes in the cytoskeleton of the host cells, which may in part explain the enhanced virulence of CagA+ H. pylori strains (11). Blaser et al. (10) showed that infection with CagA+ H. pylori was associated with a higher risk of gastric cancer, especially intestinal type affecting the distal stomach, than CagA- H. pylori infection.
The p53 gene, the most extensively studied tumor suppressor gene, is frequently mutated in various types of human cancer (16,17). The majority of p53 mutations are single base pair changes (i.e. point mutations), which result in amino acid substitutions or truncated forms of the p53 protein, and are widely distributed throughout the evolutionarily conserved regions of the gene (16,17). Examination of p53 mutations in human cancer compiled from published reports (18) has provided insights about the association between particular carcinogens and characteristic patterns of mutation (16,17,19). For example, some mutational hotspots shared by multiple cancer sites represent transition mutations at CpG dinucleotides, which are considered an endogenous process resulting from spontaneous deamination of 5-methylcytosine to thymine (20,21). In contrast, high frequency of G to T transversion mutations in lung cancer reflects the formation of DNA adducts caused by carcinogens in cigarette smoke (16,19). Even among cancers of the same organ, the mutational spectrum of the p53 gene could present different patterns by histologic subtype or risk factors involved (22,23).
In this study, we investigated whether the prevalence of p53 mutation in gastric adenocarcinoma differed between subjects with and without infection with CagA+ H. pylori strains. We also examined a relation of histologic type and anatomic subsite of tumors to the frequency of p53 mutation.
Materials and methods
Study subjects
Study subjects were selected from among the members of the Kaiser Permanente Medical Care Program (KPMCP) in Northern California who had participated in the multi-phasic health check up (MHC) between 1964 and 1969 (24) and provided sera for frozen storage. The roster of the MHC participants was linked to a cancer registry database, and 201 subjects who had had a first diagnosis of stomach cancer (ICD-9 code = 152) between the MHC enrollment and June 1996 were identified. A pathologist reviewed pathology reports and hematoxylin and eosin (H&E) slides to verify the cancer diagnosis. Subjects with histologically confirmed adenocarcinoma of the stomach, including cancer of the GEJ and gastric cardia (GEJ/cardia), who had sufficient amount of primary tumor tissue and pre-diagnostic serum available for laboratory analyses were eligible for the study.
Of the 201 subjects screened, 96 patients were excluded from the study because: there was no or insufficient gastric tumor tissue available for laboratory analyses or histologic review (48 subjects); they did not have gastric adenocarcinoma (35 subjects); there was no remaining serum (one subject) or reproducible assay results could not be obtained for the p53 gene mutational analysis (12 subjects). The remaining 105 subjects were included in the present analysis. The subjects included in the analysis were younger at diagnosis than those who would have been eligible if sufficient tumor tissue and reproducible p53 assay results had been available (mean age 70.5 versus 74.2 years; P = 0.053, t-test). There was no difference in p53 data availability by sex, race, CagA serology or tumor histologic type.
The study protocol was approved by the institutional review boards at Stanford University and the Kaiser Foundation Research Institute.
Histopathologic assessment of tumor
Two pathologists, blinded to each other's assessment, reviewed H&E slides containing gastric adenocarcinoma for each eligible subject. For each tumor, pathologists were instructed to identify a typediffuse type (signet-cell carcinoma or not otherwise specified), intestinal type (tubular, papillary or mucinous carcinoma, or not otherwise specified) or other (undifferentiated or borderline) (25). Anatomic subsite of each tumor was determined based on the structures of gastric glands surrounding the tumor or the description in pathology reports. Discordance in histologic classification (26) and anatomic subsite determination between two pathologists were resolved by a joint review.
Serological analysis for H. pylori and CagA
For each subject, an aliquot of serum was retrieved from the KPMCP-MHC serum bank that had been catalogued and maintained at the Orentreich Foundation for the Advancement of Science (Cold Spring-on-Hudson, NY). Serum specimens had been obtained 231 (median 20) years before stomach cancer diagnosis. Serological analysis was performed by the enzyme-linked immunosorbent assays (ELISAs) for H. pylori (27,28) and the CagA protein (29) as described previously. The ELISA for H. pylori was 90% sensitive and 100% specific based on 58 control samples with biopsy-proven infection status (28). The ELISA for CagA was 100% sensitive and specific in 26 control samples (29).
Mutational analysis of the p53 gene
DNA was isolated from 10 µm thick unstained sections of formalinfixed, paraffin-embedded tissue specimens, microdissected with a sterile scalpel to enrich for neoplastic cells. Microtome blades and work area were cleaned between blocks to avoid cross-contamination. Areas of the tissues where tumor cells were most abundant were chosen for microdissection by reviewing corresponding H&E slides that were used as a guide. Dissected tissues were incubated in 530 µl of 10 mM TrisHCl, 5 mM EDTA (pH 8.0) and 1 µg/µl proteinase K overnight at 56°C. Proteinase K was inactivated by heating samples in a boiling water bath for 510 min.
Extracted DNA was subjected to mutational analysis for exons 58 of the p53 gene, using the polymerase chain reactionsingle-strand conformation polymorphism (PCRSSCP) method followed by direct DNA sequencing. Exons 58 cover most of the evolutionarily conserved domain of the p53 gene, where somatic mutations in tumors have predominantly been found (17). PCR conditions and oligonucleotide primer sequences have been described previously (23,30). For all PCRs, a reagent cocktail was prepared in a laminar flow hood dedicated to pre-PCR work, using aerosol-resistant pipette tips to avoid cross-contamination. SSCP analysis was performed as described previously (31), and polyacrylamide gels were autoradiographed at room temperature without an intensifying screen. Each SSCP run included five control samples: one no-DNA control with water in place of DNA template and two each of wild-type and mutant controls whose DNA sequences had been verified previously. We ran PCRSSCP for all the samples in duplicates to reduce the possibility of false positive results due to polymerase errors or contamination. Those samples that consistently showed aberrant patterns in replicate PCRSSCP assays were subjected to direct DNA sequencing. Non-labeled PCR products were resolved on 2% agarose gels and isolated using the Mermaid oligonucleotide cleaning kit (Bio 101, La Jolla, CA). Sequencing of gel-isolated DNA was performed in both directions (i.e. for both strands) with the oligonucleotide primers used in the second round of PCRs, using the Thermo Sequenase cycle sequencing kit (USB, Cleveland, OH) according to the manufacturer's instructions.
Immunohistochemical analysis of p53 protein
Four-micrometer-thick tissue sections from the same tumor blocks as those used for mutational analysis were analyzed immunohistochemically to localize p53 protein. Following deparaffinization, rehydration and blocking of endogenous peroxidase activity with 3% H2O2 in distilled water, the tissue was incubated in 10 mM citric acid monohydrate (pH 6.0) in a microwave oven (1000 W) for a total of 15 min with periodical refills of citrate buffer. Subsequently, the tissue was rinsed with phosphate-buffered saline (PBS) and treated sequentially with three reagents: (i) DO-7 anti-p53 antibody (Dako, Carpinteria, CA) in 1:150 dilution; (ii) biotin-conjugated goat anti-mouse IgG (Jackson Immuno Research Laboratories, West Grove, PA) in 1:400 dilution; and (iii) streptavidin-conjugated horseradish peroxidase (Jackson Immuno Research Laboratories) in 1:400 dilution. Slides were rinsed with PBS twice after each reagent was applied. Finally, the site of immunoprecipitate formation was detected by applying diaminobenzidine and counterstaining with hematoxylin.
Each batch of p53 immunohistochemistry (IHC) included a mutant control (stomach cancer tissue with a mutation confirmed by DNA sequencing) and a negative control (tonsil tissue with no DO-7 antibody added). A single pathologist reviewed all the IHC slides. Each slide was scored for percentage of tumor cells with positive nuclear staining. For the purpose of the present analysis, positive IHC was defined as 5% of tumor cells with nuclear staining.
Statistical analysis
Statistical analysis was conducted using the SAS statistical package (release 8.1; Cary, NC). All P-values reported are two tailed.
Mean age at diagnosis was compared between the two anatomic subsites, distal stomach (fundus, body and antrum) versus GEJ/cardia, by t-test (32). Distributions of sex, race, CagA serology and tumor histologic type were compared between the two anatomic subsites by chi-square test (32).
An association of CagA serology with p53 mutation, while adjusting for tumor histologic type and other potential confounders, was examined by logistic regression analysis with the use of the LOGISTIC procedure (33).
Results
Of the 105 subjects included in the present analysis, 81 (77%) had cancer in the distal stomach (fundus, body and antrum) and 24 (23%) in the GEJ/cardia. Age at diagnosis was not statistically significantly different between the distal and GEJ/cardia groups (mean 71.3 versus 68.0 years; P = 0.20, t-test). The male to female ratio was higher for GEJ/cardia (5.0) than for distal stomach (2.9), although the difference was not statistically significant (P = 0.35, chi-squared test). Cancer of the GEJ/cardia was more prevalent in whites (21/77 or 27%) than in all the other races combined (3/28 or 11%) (P = 0.074, chi-squared test). The GEJ/cardia group had lower prevalence of CagA seropositivity (25% versus 75%; P < 0.001, chi-squared test) and fewer diffuse-type tumors (17 versus 33%; P = 0.12, chi-square test) than did the distal stomach group.
Twenty-nine (28%) tumors (20 of the distal stomach and nine of the GEJ/cardia) had at least one non-synonymous p53 mutation; one tumor (case no. 278) had two distinct mutations, resulting in a total of 30 mutations identified (Table I). Five (17%) and three (10%) of the 30 mutations were transition mutations at the CpG dinucleotides in codons 175 and 248, respectively. Each of the other mutations was observed only once. Observed mutations were predominantly insertions and deletions (13 mutations, 43%) and transition mutations at CpG dinucleotides (10 mutations, 33%). Positive staining for p53 protein by IHC was observed in 57 (54%) of the 105 tumors examined in this study (data not shown). When the mutational analysis results were considered as a gold standard, sensitivity and specificity of p53 IHC were 72% and 53%, respectively.
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As CagA serology is correlated with tumor histologic type and anatomic subsite, we examined the association of p53 mutation with CagA serology, histologic type and anatomic subsite by mutually adjusting for the variables in addition to the adjustment for age at diagnosis and sex of subjects (Table III). Tumors from CagA+ subjects were significantly more likely to have p53 mutation than tumors from CagA- subjects [OR = 3.72; 95% confidence interval (CI), 1.0613.07]. Tumors of intestinal and other types were more likely to have p53 mutation than diffuse-type tumors (OR = 3.56; 95% CI, 1.0612.01). GEJ/cardia tumors were more likely to have p53 mutation (OR = 2.93; 95% CI, 0.8310.35), although the association was not statistically significant.
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As of June 2000, the p53 database of the International Agency for Research on Cancer (IARC) (18) listed 378 gastric cancer cases with point mutations in the p53 gene. Of these tumors, each of the following codons of the p53 gene was mutated in >10 tumors: codons 175, 213, 245, 248, 273 and 282. Mutations in all but one (codon 245) of these six hotspots were transition mutations at CpG dinucleotides. Notably, mutation at one of these hotspots (codon 248) can be induced in vitro by reactive oxygen species and a nitric oxide-releasing compound (34). Increased production of nitric oxide caused by chronic inflammation and subsequent oxidative damage to the gastric epithelium is one of the hypothesized mechanisms of H. pylori-induced gastric carcinogenesis (2,35). Increased expression of the inducible isoform of nitric oxide synthase is positively correlated with the frequency of G:C to A:T transition mutations at CpG sites of the p53 gene in colon cancer (35). CpG mutations can also result from a spontaneous deamination of 5-methylcytosine at CpG sites and, along with deletion/insertion mutations, consist of the principal mutation types among somatic p53 mutations in various cancer sites as well as germline p53 mutations in Li-Fraumeni syndrome patients (17,21).
Overall, p53 mutation was found in 25% of gastric cancers examined in this study. This prevalence falls within a wide range of p53 mutation frequency [8% (36) to 65% (37)] in gastric adenocarcinoma from previous studies that used similar methods of mutation detection. A CpG mutation at codon 175 stood out as a mutational hotspot in this study sample, accounting for five of 30 mutations identified. Insertions and deletions were more frequent in our series (43%) than in the mutations compiled in the IARC database [9% as of June 2000 or 16% in a summary by Greenblatt et al. (16) published in 1994]. As the sources and selection criteria of tumors, even for the same anatomic site (e.g. stomach) and major histologic type (e.g. adenocarcinoma), could vary substantially across the reports represented in the IARC database, we cannot judge whether the relatively high prevalence of deletions/insertions observed in the present study has any biological implications.
As CagA+ H. pylori strains induce particularly severe inflammation in the gastric mucosa (8,9), we hypothesized that gastric tumors from subjects infected with CagA+ H. pylori have a higher prevalence of p53 mutation than tumors from subjects with no such infection. We found that gastric tumors from CagA+ subjects were 3.7 times more likely to harbor p53 mutation than tumors from CagA- subjects after adjustment for histologic type and anatomic subsite of cancer as well as age at diagnosis and sex of subjects. To our knowledge, only one study to date has reported on the prevalence of p53 mutation in gastric adenocarcinoma in relation to CagA status (38). In that study of 64 patients by Deguchi et al. (38), CagA status was determined based on gastric biopsies obtained at the time of cancer diagnosis as opposed to retrospective determination using pre-diagnostic sera as carried out in the present study. None the less, the result of the present study is consistent with the finding of Deguchi et al. (38) that p53 mutation was more prevalent in tumors from CagA+ subjects than those from CagA- subjects (30% versus 7%, P = 0.033).
The availability of pre-diagnostic sera helps establish a temporal association of the `exposure' and `outcome' events. In addition, antibody titers for H. pylori and CagA tend to decline as the gastric epithelium undergoes histopathological changes to form intestinal metaplasia and stomach cancer (3941), leading to potential misclassification of subjects with respect to the history of infection. On the other hand, a long interval between serum sample collection and stomach cancer diagnosis leaves a possibility that some initially H. pylori- subjects might have acquired the infection during the follow-up period. However, the majority of H. pylori infections seem to be acquired during childhood (2,42). Subjects in the present study were 21 years of age or older at the time of serum collection. Furthermore, the mean interval between serum collection and stomach cancer diagnosis in this study was similar regardless of CagA serology and p53 mutation status: 20.6 years for CagA-, p53 mutation- (n = 30); 22.8 years for CagA-, p53 mutation+ (n = 8); 20.5 years for CagA+, p53 mutation- (n = 46) and 21.9 years for CagA+, p53 mutation+ (n = 21).
We observed that GEJ/cardia cancer was more likely to have p53 mutation even after adjustment for CagA serology and tumor histology, although the association was not statistically significant. The higher prevalence of p53 mutation in GEJ/cardia cancer than in distal stomach cancer was also found in another study (43). Gastroesophageal reflux disease is a major risk factor for intestinal metaplasia (Barrett esophagus) and cancer of the GEJ/cardia (44,45) whereas CagA+ H. pylori infection may be inversely associated with the risk of GEJ/cardia cancer (46). The difference in p53 mutation frequency between cancers of the GEJ/cardia and distal stomach may reflect the differences in the main causes of epithelial damage for the two anatomic subsites.
p53 mutation was less frequent in diffuse-type tumors than in tumors of intestinal and other types, which is consistent with previous reports (43,4750). The marked decline in the age-standardized incidence rates of gastric adenocarcinoma over the last few decades are mainly due to the change in intestinal-type tumor occurrence (4). This descriptive epidemiologic observation supports the hypothesis that exogenous risk factors play a larger role in the carcinogenesis of intestinal-type tumors than that of diffuse-type tumors (5), which may explain the higher prevalence of somatic p53 mutation in intestinal-type tumors observed in this study. In contrast, germline mutations in the E-cadherin gene have been linked to diffuse-type, but not intestinal-type, gastric cancer (51). It should be noted, however, that the sensitivity of p53 mutation detection could be lower for diffuse-type cancer than for intestinal-type cancer because of the relatively small number of malignant cells in diffuse-type tumors (52). IHC, an alternative way of evaluating p53 abnormalities, may be less susceptible to this problem as a pathologist reviewing immunostained sections can distinguish tumor cells from non-malignant cells.
The p53 protein with a prolonged half-life detected by IHC in archival tissue specimens is often, but not always, associated with mutations in the p53 gene. The discrepancy between IHC and mutational analysis, particularly the low specificity, observed in this study could be due to mutations outside exons 58, frameshift mutations leading to truncated protein that may affect the presentation of epitopes and/or overexpression of p53 protein caused by mechanisms other than genetic mutation. For instance, seven (78%) of the nine tumors with frameshift mutation in the p53 gene were negative by IHC whereas only one (5%) of the 20 tumors with other types of mutation were IHC negative (Table I). Although it is possible that we missed p53 mutation in some tumors, previous studies that surveyed wider regions of the coding sequence did not necessarily find a higher prevalence of p53 mutation (43,5355).
In conclusion, we found p53 mutation was more prevalent in tumors from CagA+ subjects than in tumors from CagA- subjects. The latter group included both H. pylori- and H.pylory+/CagA- subjects, which could not be examined separately due to the small sample size of those subgroups. Nevertheless, our findings provide further support for heterogeneity of carcinogenesis by H. pylori phenotype marked by CagA serology.
Notes
8 To whom correspondence should be addressed Email: ashibata{at}stanford.edu
Acknowledgments
The authors thank Bonnie Bell, Lucy Bonds, Bill Frank, Eva Pfendt, Shufang Yang, and Donna Wells for technical assistance; Dr Gary Friedman for access to databases and other resources at the Kaiser Permanente Medical Care Program (KPMCP); and pathology departments of the KPMCP in Northern California for providing archival tissue specimens. This work was supported in part by Institutional Research Grant #3236 from the American Cancer Society and Public Health Service grants R01CA73011 and R35CA49761 from the National Cancer Institute, the National Institutes of Health.
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