Japanese Journal of Clinical Oncology 30:423-428 (2000)
© 2000 Foundation for Promotion of Cancer Research
Association Between p53 Immunostaining and Cigarette Smoking in Squamous Cell Carcinoma of the Esophagus
1Department of Surgery II, 2Department of Pathology II and 3Vice President, Kochi Medical School, Kochi, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Background: It is generally accepted that cigarette smoking is closely associated with esophageal squamous cell carcinoma. This study investigated the molecular targets of cigarette smoke in carcinogenesis of the esophagus.
Methods: Seventy-four patients with esophageal squamous cell carcinoma (SCC) were grouped according to daily cigarette consumption: heavy smoking group (group H) (n = 26), moderate smoking group (group M) (n = 39) and non-smoking group (group N) (n = 9). We compared p53 and retinoblastoma (RB) expression among the three groups by immunohistochemistry. In addition, fresh tumor tissues from 30 smokers with esophageal SCC were tested for p53 mutations in exons 5–8 by direct sequencing.
Results: Staining for the p53 product was positive in 65.4% of group H, 38.5% of group M and 44.4% of group N. The frequency of positive staining in the group H was significantly higher than in group M (p = 0.033) and in group M + group N (p = 0.034). The difference with respect to the frequency of overexpression of RB was not significant. The patterns of p53 base-pair mutations in direct sequencing study were of five types, most commonly G:C to T:A transversion (35.3%).
Conclusions: Our study suggests that one of the molecular targets of cigarette smoke is the p53 gene. The pattern of p53 point mutations involved a wide range of base-pair changes.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is generally accepted that cigarette smoking is closely associated with esophageal squamous cell carcinoma (SCC) (1,2). However, the molecular targets of cigarette smoke have not been firmly identified. For head and neck SCC, another neoplasm related to smoking, Brennan et al. (3) reported that a history of tobacco consumption is associated with a high frequency of p53 mutations. The p53 and retinoblastoma (RB) tumor suppressor genes, which encode nuclear phosphoproteins that play a regulatory role in normal cell growth, have recently been shown to be crucial in the pathogenesis of human esophageal cancer (4). To clarify the molecular targets of cigarette smoke, we allocated patients with primary esophageal SCC to three groups according to daily cigarette consumption and compared p53 and RB expression by immunohistochemistry among the groups. In addition, p53 mutations were examined in 30 smokers by direct DNA sequencing.
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PATIENTS AND METHODS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A total of 74 patients (63 men and 11 women), who underwent esophagectomy for esophageal SCC at our hospital between March 1982 and April 1996 were evaluated retrospectively. They were grouped according to daily cigarette consumption: a heavy smoking group (group H), with daily consumption of over 21 cigarettes for more than 10 years (n = 26); a moderate smoking group (group M), with daily consumption of between one and 20 cigarettes for more than 10 years (n = 39); and a non-smoking group (group N) (n = 9). The clinicopathological features of the patients and the frequency of overexpression of cancer-related genes, including p53 and RB, were compared among the three groups. From 30 patients in groups M and H who underwent esophagectomy for esophageal SCC between May 1993 and April 1998, tumor residues were snap-frozen and stored at –80°C. DNA was extracted as described previously (5) from the surgical specimens. The location of the tumor within the esophagus and the depth of tumor invasion were determined according to the TNM classification (6). Information on etiological factors, including tobacco and alcohol consumption and a family history of cancer, was obtained for each patient from hospital charts. Alcohol consumption was converted into grams of ethanol based on the content of each type of beverage.
Immunohistochemistry
Sections 5 µm thick from archival formalin-fixed paraffin-embedded tissues were placed on poly-L-lysine-coated slides (Sigma Chemical, St. Louis, MO) for immunohistochemistry study. The expression of p53 and RB proteins were assessed by immunohistochemistry using an anti-p53 monoclonal antibody (DO-7, Dako, Kyoto, Japan; diluted 1:30) and an anti-human RB monoclonal antibody (3H3, MBL, Nagoya, Japan; dilution 1:40). After blocking of endogenous peroxidase activity, the sections were washed in phosphate-buffered saline (PBS; pH 7.4). The heat-induced antigen retrieval technique was used for each immunostaining. The sections were heated in a pressure cooker with 10 mM sodium citrate buffer (pH 6.0) for 12 min at 132°C. Deparaffinized sections were pretreated with normal goat serum for 30 min and incubated with each antibody at 4°C for 24 h. After washing with 0.1 M PBS (pH 7.4), the streptavidin–biotin complex (ABC) procedure was performed using a streptavidin–biotin complex peroxidase kit (DAKO LSAB kit, Dakopatts, Kyoto, Japan). The sections were briefly counterstained with methyl green before mounting. Control sections of known positive cases of esophageal SCC were included in each run and a negative control was carried out by omitting the primary antibody.
Nuclear staining was considered positive if the chromogen was detected in at least 5% of all nuclei within a microscopic field (5,7). The expression of RB protein in a tumor was considered to be negative when positive nuclear staining was observed in immediately adjacent non-neoplastic cells, but not in the tumor cell itself (5).
Direct Sequence Analysis for p53
Our study concentrated on exons 5–8 of the gene, since previous studies had demonstrated that these exons harbor the most inactivating mutations in many diverse types of human tumors. Direct DNA sequencing was performed using a procedure described previously (8). The sequences of the primers used for polymerase chain reaction (PCR) amplifications were as follows: exon 5 (forward) TTCCTCTTCCTGCAGTACTC, (reverse) GCTCCAGCTGCTCACCATCG; exon 6 (forward) CACTGATTGCTCTTAGGTCTG, (reverse) AGTTGCAAACCAGACCTCAG; exon 7 (forward) GTGTTGTCTCCTAGGTTGGC, (reverse) CAAGTGGCTCCTGACCTGGAG; exon 8 (forward) CCTATCCTGAGTAGTGGTAATC, (reverse) GCTCTGCTTGCTTACCTCGC.
Differences among the three groups were analyzed using the chi-squared test with Fisher’s exact probability test and by Student’s t-test.
Because of the small size of group N, we compared the degrees of p53 and RB expression by immunohistochemistry among the three groups and between group H and group M + group N.
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RESULTS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Clinicopathological Features
The average daily cigarette consumption in groups H and M was 40.7 ± 16.5 and 17.4 ± 4.3, respectively. There was a significant difference between the two groups (p < 0.001). Although the cigarette-year in group H was significantly shorter than in group M (p = 0.009), the tobacco index in group H was significantly higher than in group M (p < 0.001). The average age of patients in group H was significantly lower than those in groups M (p < 0.001) and N (p = 0.006). The number of women in group N was significantly higher than those in the smoking groups (p < 0.001). In the smoking groups, tumors occurred most frequently in the middle thoracic esophagus. Patients in group N, on the other hand, developed tumors most frequently in the lower thoracic region. The difference in tumor location between the smoking groups and group N was significant (group H vs group N, p = 0.007; group M vs group N, p = 0.007). The differences in the histological features, the depth of tumor invasion (pTNM classification) and lymph node metastasis among the three groups were not significant (Table 1). Ten (38.5%) of the patients in group H, 21 (53.8%) in group M and six (66.7%) in group N had third-degree or closer blood relatives with cancers. The difference in the incidence among the three groups was not significant.
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Immunohistochemistry
With respect to the anti-p53 antibody, 65.4% of the 26 tumors in group H, 38.5% of the 39 tumors in group M and 44.4% of the nine tumors in group N exhibited positive nuclear staining (Fig. 1). The frequency of overexpression of p53 in group H was significantly higher than in group M (p = 0.033) and group M + group N (p = 0.034) (Table 2). The differences among the three groups with respect to the overexpression frequency of RB were not significant (Fig. 2 and Table 2).
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p53 Gene Mutation
The p53 gene was sequenced in tumor specimens from 30 moderate and heavy smokers with esophageal SCC. As shown in Table 3, 20 mutations in the p53 gene were found in 19 of the 30 cases. Seventeen point mutations were detected in 16 cases, including nine in exon 5, one in exon 6, four in exon 7 and three in exon 8. The patterns of p53 base-pair mutations were of five types, involving six cases of G:C
T:A (35.3%), four of G:CA:T (23.5%), three of A:TG:C (17.6%), three of A:TT:A (17.6%) and one of G:CC:G (5.9%). The 17 point mutations included 13 missense mutations (all of which were positive for p53 protein overexpression), three nonsense mutations and one silent mutation. Of the three cases showing nonsense mutation, one was positive for p53 staining and the others were negative. Of the 11 samples with no alteration of the p53 gene, four were not stained for p53, while the remaining seven samples exhibited positive staining for p53.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Epidemiological studies have revealed that cigarette smoking is positively associated with esophageal SCC. However, very little is known about the effects of cigarette smoking on the molecular events associated with esophageal SCC. Therefore, we evaluated the relationship between cigarette consumption and overexpression of oncological genes associated with esophageal SCC.
The average age of the patients in group H was significantly lower than that in group M. Group H and group M did not differ significantly in male-to-female ratio, the depth of tumor invasion, tumor location, histology of the tumor, lymph node metastasis, the number of drinkers, the average daily alcohol consumption or the number of patients with a family history of cancer. It is therefore likely that the frequency of cigarette smoking is associated with a low age at onset in group H.
The p53 gene, encoding a 53 kDa nuclear phosphoprotein, is a representative tumor suppressor gene that is a crucial regulator of cell growth, differentiation and apoptosis through its actions in cell-cycle checkpoint control (9). Immunohistochemical positivity for p53 protein is in general thought to reflect point mutations of p53 genes in tumor cells, although it is not always synonymous with p53 mutations. In esophageal SCC, the frequency of p53 immunopositivity is reported to be 30–85% (5,10–14). In the present study, the overexpression frequency of p53 was 48.0% (36/75). Lam et al. (15) reviewed 70 cases of esophageal SCC and showed that tobacco use had no significant relationship with positivity or staining intensity for p53, as detected by immunohistochemistry. However, in their study the patients were classified as smokers when they had smoked one or more packs of cigarettes a day for over 2 years. Montesano et al. (16) demonstrated that tobacco smokers with esophageal SCC were much more likely to have p53 mutations than non-smokers with esophageal SCC and the frequency of p53 mutations increased with the dose (number of cigarettes smoked per day). In the present study, smokers who had smoked for more than 10 years were grouped according to their daily cigarette consumption. We found that the frequency of p53 overexpression in group H was significantly higher than those in group M and in group M + group N.
Recent studies have indicated that p53 gene alteration as well as p53 protein accumulation are frequently detected in dysplastic or precancerous lesions adjacent to SCC of the esophagus (11,17–19). Thus, such a p53 alteration may occur as an early event in the tumorigenesis of esophageal SCC. In the present study, therefore, a low age at tumor onset in group H may be associated with a high frequency of p53 overexpression.
In head and neck SCC, a wide range of p53 mutations have been found, most commonly G:CA:T, G:CT:A and A:TG:C (3). In our patients with SCC of the esophagus who used tobacco, the pattern of p53 point mutations was almost the same as that in head and neck SCC. Several reports have discussed the high incidence of the association of separate primary tumors in the head and neck region and the esophagus (2,20–22). The head and neck region is adjacent to the esophagus. The mucosae in each are composed of squamous epithelia and are similarly exposed to environmental carcinogens. It is therefore reasonable that the wide range of patterns of p53 point mutations detected in our cases of esophageal SCC is similar to that in head and neck SCC.
Carcinogens may leave unique ‘fingerprints’ in the form of specific mutations that cause the initiation or progression of cancer (23,24). With respect to cigarettes, Puisieux et al. (25) suggested that benzo[a]pyrene in tobacco smoke specifically causes G:CT:A mutations in the p53 gene in lung cancer. Mutations of the p53 gene in patients with bladder cancer who smoked typically consisted of G:CC:G and A:TG:C (26,27). These mutations may result from the aromatic amines and N-[4-(5-nitro-2-furyl)-2-thiaxolyl]formamide, both of which are present at increased levels in urothelial cells of cigarette smokers. The different base-pair changes of p53 point mutations in the present study, therefore, suggest that the smoked cigarette produces various kinds of carcinogens, although further accumulation of cases and studies will be necessary.
Normal RB protein exhibits positive nuclear staining by immunohistochemistry and it has been established that the finding of a negative RB protein expression pattern permits a fairly good estimation of the mutation frequency in the RB gene (28,29). In the present study, there was no significant difference in the frequency of cases showing negative RB protein among the three groups and between group H and group M + group N. Therefore, the RB protein may not be a site of genetic damage caused by cigarette smoking.
In group N, the proportion of non-smoking, non-drinking women was significantly high compared with the smoking groups. Additionally, although tumors in the smoking groups most frequently occurred in the middle thoracic esophagus, those in group N most frequently developed in the lower thoracic esophagus. These findings suggest that other factors may be associated with carcinogenesis in non-smoking and non-drinking patients. It will be important to examine a larger number of cases to evaluate carcinogenesis in non-smoking and non-drinking patients.
In conclusion, the results of this study indicate that one of the molecular targets of cigarette smoke is the p53 gene and that damage to this gene depends on the number of cigarettes smoked per day. Furthermore, the pattern of p53 point mutations in SCC of the esophagus shows a wide spectrum of base-pair changes.
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FOOTNOTES |
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+ For reprints and all correspondence: Shunji Mizobuchi, Department of Surgery II, Kochi Medical School, Kohasu, Okohcho, Nankoku, Kochi 783-8505, Japan. E-mail: mizoshun@kochi-ms.ac.jp
Abbreviations: SCC, squamous cell carcinoma; RB, retinoblastoma
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Received April 20, 2000; accepted July 24, 2000.
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