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The relation of alcohol consumption and cigarette smoking to the multiple occurrence of esophageal dysplasia and squamous cell carcinoma
The relation of alcohol consumption and cigarette smoking to the multiple occurrence of esophageal dysplasia and squamous cell carcinoma Mitsuhiro Miyazaki, MD, Shinji Ohno, MD, Motonori Futatsugi, MD, Hiroshi Saeki, MD, Takefumi Ohga, MD, and Masayuki Watanabe, MD, Fukuoka, Japan
Background. The unique pathologic features of esophageal tumors in patients with esophageal cancer includes the presence of multiple occurrence within the esophagus. The aim of this study is to clarify the molecular mechanism of carcinogenesis of multiple esophageal squamous cell carcinomas in the Japanese. Methods. We studied the relationship between the incidence of patients with multiple carcinomas and the coexistence of dysplasia lesions with p53 protein accumulation, alcohol consumption, and cigarette smoking. Among 76 cancer lesions and 60 cases of dysplasia, p53 accumulation was studied by means of immunohistochemical analysis. Results. The incidence of patients with multiple carcinomas in the high-risk group was 33%, and the incidence of patients with a coexistence of dysplasia in the high-risk group was 67%. The incidence of patients with multiple carcinomas or the coexistence of dysplasia in the high-risk group was much higher than that of the middle-risk and low-risk groups (P < .0001 and P = .04, respectively). The average number of abnormal epitheliums, such as cancer and dysplasia, in the high-risk group was 3.2 ± 2.1. The average number of abnormal epitheliums was much higher than that of the other groups (P = .02). For carcinoma lesions, the incidence of lesions with a positive p53 protein accumulation in the high-risk group was 91%. Regarding dysplasia lesions, the incidence of lesions with a positive p53 protein accumulation in the high-risk group was 80%. The incidence of both cancer and dysplasia lesions with a positive p53 protein accumulation in the high-risk group was higher than that of the other groups. Conclusions. The pattern of p53 accumulation in dysplasia in the high-risk group was closely similar to that in cancer of the high-risk group. These findings support the concept of field carciogenesis of the esophagus. (Surgery 2002;131:S7-13.) From the Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
IN PATIENTS WITH SQUAMOUS cell carcinoma of the esophagus, one of the most unique pathologic features of the tumors is the presence of multiple occurrence within the esophagus. We found the incidence of this multiplicity to be 25.6% in patients not given preoperative radiation treatment.1 It is important to study the carcinogenesis of multiple occurrences in esophageal squamous cell carcinoma. Epidemiologic studies revealed that alcohol consumption and cigarette smoking are the major risk factors of squamous cell carcinoma of the Reprint requests: Mitsuhiro Miyazaki, MD, Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 8128582, Japan. Copyright © 2002 by Mosby, Inc. 0039-6060/2002/$35.00 + 0 11/0/119288 doi:10.1067/msy.2002.119288
esophagus. In Japan the estimated world age-standardized annual incidence of esophageal cancer is 8.9/100,000 for men and 1.5/100,000 for women. Alcohol consumption and cigarette smoking are considered to be the major risk factors for squamous cell carcinoma of the esophagus in Japanese persons living in Japan. Alcohol consumption is associated with an increased risk of esophageal cancer, and the amount of alcohol consumed was found to be the main risk determinant.2 A statistically significant dose-response relationship was noted between cigarette smoking and the mortality rate for carcinomas of the esophagus in men.3 Some studies, such as immunohistochemical detection of proliferation cell nuclear antigen for resected specimens, demonstrated that the biologic nature of the dysplasia might well be as serious a lesion as cancer itself.4 Carcinomas coexistent with dysplasia have been reported to be more frequentSURGERY S7
S8 Miyazaki et al
Fig 1. Distribution of the patients in this study on the basis of drinking index and pack-years.
ly seen in less advanced esophageal cancer.5 As a result, dysplasia is considered to be a good subject to study the cancerization of squamous cell carcinoma of the esophagus. We studied dysplasias in the patients with squamous cell carcinoma of the esophagus limited to the submucosal layer. p53 is one of the well-known oncosuppressor genes related to the tumor carcinogenesis in esophageal squamous cell carcinoma.6 Immunohistochemical analyses with specific antibodies to p53 showed that an abnormal accumulation of p53 protein was recognized at precancerous stages, such as basal cell hyperplasia and dysplasia of the esophagus, suggesting that p53 mutations are indeed important events in the early stage of esophageal squamous cell cancerization.7-9 The expression of p53 protein in esophageal squamous cell carcinoma tissue and dysplasia tissue taken from Japanese patients was immunohistochemically investigated, and the relationship between the incidence of patients with multiple cancer lesions and dysplasia and p53 protein accumulation and the patient’s history of alcohol consumption and cigarette smoking was analyzed. The aim of our study was to elucidate the molecular mechanism involved in the occurrence of multiple esophageal dysplasia and squamous cell carcinoma. MATERIALS AND METHODS Patients. Our study was based on 68 patients with primary esophageal squamous cell carcinoma surgically treated in our institute from 1989 to 1998. The main tumors of all patients were limited to the submucosal layer. In addition, none of the
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patients had been given either preoperative irradiation or chemotherapy. Microscopic sections of the whole resected esophagus were prepared from step-sectioned blocks measuring 5 mm in width and were stained with hematoxylin and eosin. The differentiation of squamous cell carcinoma and the TNM stage were determined on the basis of histologic evaluations. Regarding the histologic diagnosis of squamous epithelial dysplasia, we used the criteria defined by the World Health Organization’s International Histological Classification of Tumors in 1990, according to which the nuclei of the lesion are enlarged and hyperchromatic while showing an increased mitotic activity. In the case of mild dysplasia, cells with such atypical nuclei were limited to the basal layer. With the increasing grade of dysplasia, there was a progressive increase of the cells with atypical nuclei until the entire thickness of the epithelium was replaced. Then moderate dysplasia was diagnosed when the zone with atypical cells expanded up to one half of the epithelium. Severe dysplasia was diagnosed when the zone with atypical cells expanded up to three quarters of the epithelium. In addition, intraepithelial carcinoma was diagnosed when an epithelium was replaced with atypical cells either completely or almost completely without any invasive growth. All sections with squamous cell carcinoma and dysplasia were selected for this study. The patients were retrospectively evaluated from the medical records of the Registry of Esophageal Cancer at the Second Department of Surgery, Kyushu University. To define the use of alcohol and cigarettes, we used the same questionnaire for all patients, as described previously.10,11 One drink of alcohol corresponded to 180 mL of sake (the most popular rice-wine alcoholic beverage in Japan), 120 mL of white liquor (shochu), 70 mL of whisky, and 720 mL of beer. A drinking index of the accumulated amount of ethanol was defined as the number of drinks per week times the number of years of drinking. For example, those with a history of drinking 360 mL of sake per day for 30 years had a score of 420 on the drinking index. In terms of the accumulated amount of smoking, “pack-years” was defined as follows: (Number of cigarettes per day/20) × Number of years of smoking.
For example, a patient with a history of smoking 20 cigarettes per day for 30 years was scored as at 30 pack-years. All patients were distributed on the basis of drinking index and pack-years, as shown in Fig 1. Immunohistochemistry. p53 immunohistochemical staining was performed on formalin-fixed,
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Fig 2. Incidence of patients with multiple cancer lesions or the coexistence of dysplasia for each group. The incidence of the patients with multiple cancer lesions was significantly higher than that of the other groups (P < .0001). The incidence of the patients with the coexistence of dysplasia was significantly higher than that of the other groups (P = .04).
paraffin-embedded esophageal tissue with squamous cell carcinoma or dysplasia from all patients. The sections were dewaxed in xylene and rehydrated with ethanol and then microwaved at 95°C in phosphate-buffered saline for 20 minutes. These sections were incubated with 0.3% hydrogen peroxidase in methanol to block endogenous peroxidase activity. Normal rabbit serum was applied for 20 minutes to reduce any nonspecific antibody binding. A mouse monoclonal antibody against p53 (DO-7; Dako, Glostrup, Denmark) with a 1:100 dilution was incubated overnight at 4°C. Staining was detected by using the streptavidin-biotin-peroxidase complex method. p53 protein accumulation was investigated in each specimen by investigators blinded to the risk-factor data. When immunoreactive neoplasia cells accounted for over 10%, a positive result was recorded. Statistical analysis. The data on the patient group were compared with either the chi-square test or the Student t test. The average number of dysplasias per one patient and p53 protein accumulation were compared in esophageal squamous cell carcinoma and dysplasia to evaluate the correlation between the risk-factor data and the incidence of the patients with multiple carcinomas or the coexistence of dysplasia. RESULTS Patient population. The clinicopathologic findings of all patients are listed in Table I. No significant difference was seen among the 3 groups (lowrisk, middle-risk, and high-risk groups) regarding sex, age, length, depth, location, and differentiation (data not shown). There were 76 carcinoma
lesions and 60 dysplasia lesions among the 68 patients. The average number of carcinoma lesions is 1.1 ± 0.4 per patient. The average number of dysplasia lesions was 0.8 ± 1.6 per patient. Relationship between the incidence of cases with multiple cancer or dysplasia and both alcohol consumption and cigarette smoking. Among the 68 patients with esophageal squamous cell carcinoma, 15 (22.1%) belonged to the high-risk group, 32 (47.0%) belonged to the middle-risk group, and 21 (30.9%) belonged to the low-risk group. Among the 68 patients with esophageal squamous cell carcinoma, 5 (7.4%) belonged to the group with multiple carcinomas, and 26 (38.2%) belonged to the group with a coexistence of dysplasia. All of the patients with multiple carcinomas belonged to the high-risk group. The incidence of the patients with multiple carcinomas in each group was 33%, 0%, and 0% in the high-risk, middle-risk, and low-risk groups, respectively. In addition, the incidence of patients with a coexistence of dysplasia in each group was 67%, 29%, and 32% in the high-risk, middle-risk, and low-risk groups, respectively. There was a significant correlation between the incidence of the patients with multiple carcinomas or the coexistence of dysplasia and the risk factors, as shown in Fig 2 (P < .0001, P = .04). Relationship between the average number of dysplasias per patient and both alcohol consumption and cigarette smoking. Among the 68 patients with esophageal squamous cell carcinoma, the average number of dysplasias per patient is 0.8 ± 1.6. In addition, the average number in each group was 1.7 ± 1.6, 0.8 ± 1.7, and 0.5 ± 1.0 in the highrisk, middle-risk, and low-risk groups, respectively,
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Fig 3. Average number of dysplasia lesions per patient with esophageal squamous cell carcinoma for each group. There is a significant difference between the average number of dysplasia lesions for the high-risk and low-risk groups (P = .01).
Table I. Clinicopathologic findings of all patients (n = 68) Sex (M/F) Age (y) Length (cm) Location Upper Middle Lower Depth Tis T1 Differentiation Well Moderate Poor Expression of multiple cancer Negative Positive Expression of dysplasia Negative Positive No. of carcinomas per patient No. of dysplasia lesions per patient Drinking index Pack-years
62/6 63.1 ± 9.3 (36-76) 3.2 ± 1.7 (1-10) 10 38 20 4 64 5 49 14 63 5 42 26 1.1 ± 0.4 (1-3) 0.8 ± 1.6 (0-8) 630 ± 634 (0-4190) 40 ± 36 (0-150)
Values in parentheses are ranges.
as shown in Fig 3. There was a significant difference regarding the average number of dysplasias per patient between the high-risk and low-risk groups (P = .01). Alcohol consumption, cigarette smoking, and p53 protein accumulation in esophageal squamous
cell carcinoma and dysplasia. p53 protein– immunoreactive cells are rare in the normal squamous epithelium. Among the 75 cancer lesions of 68 patients with esophageal squamous cell carcinoma, 46 (61%) belonged to the p53 protein accumulation–positive group and 30 (39%) to the negative group. Among the 60 dysplasia lesions of 68 patients, 38 (63%) belonged to the p53 protein accumulation–positive group and 22 (37%) belonged to the negative group. p53 protein immunoreactivity was observed in the nucleus of each cell. In most specimens in the p53 protein accumulation–positive group, the p53 protein immunoreactive neoplastic cells were diffuse throughout the carcinoma and dysplasia (Fig 4). Fig 5 shows the correlation between alcohol consumption, cigarette smoking, and p53 protein accumulation in both carcinoma and dysplasia. For the carcinoma lesions, the incidence of the lesions with positive p53 protein accumulation was 91%, 48%, and 45% for the high-risk, middle-risk, and low-risk groups, respectively. For dysplasia lesions, the incidence of lesions with positive p53 protein accumulation was 80%, 54%, and 45% for the highrisk, middle-risk, and low-risk groups, respectively. A significant correlation was seen between the incidence of the lesions with positive p53 protein accumulation and both alcohol consumption and cigarette smoking for carcinoma lesions (P = .001). For dysplasia lesions, no significant correlation was observed between the incidence of the lesions with a positive p53 protein accumulation and both alcohol consumption and cigarette smoking. The incidence of the lesions with positive p53 protein accu-
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Fig 4. Image of a cancerous lesion with a p53 protein–positive expression. In most specimens in the p53 protein accumulation–positive group, p53 protein–immunoreactive neoplastic cells were diffuse throughout the carcinoma and dysplasia, such as observed in this case.
Fig 5. Incidence of lesions with p53 protein accumulation in esophageal squamous cell carcinoma and dysplasia for each group. For the carcinoma, the incidence of lesions with p53 protein accumulation for the high-risk group was significantly higher than that of the other groups (P = .0014). For dysplasia, there is no significant difference among the 3 groups, but the incidence of the lesions with p53 accumulation tended to be higher than that of the other groups (P = .068).
mulation did tend to be higher in the high-risk group than in the other groups (P = .068). DISCUSSION p53 has come to the forefront of cancer research because it is commonly mutated in human malignant disease.12-15 The timing of p53 protein–related alterations in carcinogenesis and progression can vary with the tumor. In the case of the esophagus, p53 alterations can occur in the early stage of cancer and may thus confer a growth
advantage to preinvasive cells.7-9 In our previous study we reported that alcohol consumption and cigarette smoking were related to a high frequency of p53 protein accumulation in esophageal squamous cell carcinoma in the Japanese.11 As a result, in this study we used dysplasia of the patients with carcinoma limited to the submucosal layer that has been recognized as precancerous to investigate the relationship between such risk factors as alcohol drinking and cigarette smoking to an abnormality in p53 for carcinogenesis for multiple esophageal
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cancer. We investigated the p53 protein expression in 68 cases of esophageal squamous cell carcinoma in Japanese patients by using a highly specific antibody to p53, and we correlated the results with the patients’ history of using alcohol and cigarettes, both important causative factors in esophageal squamous cell carcinoma. To our knowledge, this is the first report to focus on a correlation between the epidemiologic risk factors and the expression of a multiple occurrence of dysplasia and cancer of the esophagus and an aberrant expression of a tumor-suppressor gene. We previously reported that heavy smoking and alcohol consumption are clearly associated with multiple occurrences of upper aerodigestive tract cancer in Japanese patients.10 The multicentric occurrence of dysplasia and squamous cell carcinoma in the same esophagus was at a certain frequency.10 In this study the same results were also recognized. These findings suggest that the multiple occurrence of dysplasia and squamous cell carcinoma might be related to alcohol consumption and cigarette smoking. Because we especially used cases with early esophageal squamous cell carcinoma in this study, the results of this study reflect the relationship between the multiple occurrence of squamous cell carcinoma and such risk factors as alcohol consumption and cigarette smoking. Our findings show that the accumulation of p53 protein in both carcinoma and dysplasia is closely associated with large amounts of both alcohol and cigarette use. In our previous study the accumulation of p53 protein was also closely related to such risk factors as heavy alcohol consumption and cigarette smoking.11 These findings suggest that the p53 protein accumulation of dysplasia might be related to such risk factors as heavy alcohol consumption and cigarette smoking regarding squamous cell carcinomas of the esophagus. The incidence of the multiple occurrence of the dysplasia and p53 accumulation in the dysplasia was higher in the high-risk group, which was similar to the finding of squamous cell carcinomas of the esophagus. As a result, the multiple carcinogenesis of the squamous cell carcinoma of the esophagus might well be related to the p53 mutation of the squamous epithelium as a result of the carcinogens supplied by heavy alcohol consumption and cigarette smoking. In normal cells p53 is expressed at a low level and has a short half-life (6 minutes in the spleen),16 and a change in molecular configuration leads to a stabilization of the protein product so that its half-life is greatly extended. Immunohistochemical screen-
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ing for elevated protein levels followed by a sequence analysis represents an efficient strategy for the elevation of the p53 mutational spectrum.17,18 In the current study p53 protein accumulation was immunohistochemically screened in 68 patients with primary esophageal squamous cell carcinoma. A sequence analysis is needed in ongoing studies to elucidate the association between the risk factors and mutation of p53 in esophageal squamous cell carcinoma. Regarding “field carcinogenesis,” this term was used to represent various aspects. There is a viewpoint of synchronous or metachronous cancer development in multiple organs, such as upper aerodigestive tract cancer.1,19,20 The multiple occurrence of carcinoma within one organ suggests the possibility of field carcinogenesis.21,22 The term “field carcinogenesis” indicates a phenomenon of almost simultaneous multicellular carcinogenesis. On the basis of our findings, the main mechanism of the multiple occurrence of esophageal squamous cell carcinoma in patients with heavy alcohol consumption and cigarette smoking may be due to field carcinogenesis. Our findings show that the pattern of p53 accumulation of the dysplasia in each group was similar to that of cancer in each group. From these findings, we conclude that the multiple carcinogenesis of esophageal squamous cell carcinoma is related to a mutation of p53 in a large area in the same esophagus by the exposure of carcinogen caused by large amounts of alcohol drinking and cigarette smoking. REFERENCES 1. Kuwano H, Morita M, Tsutsui S, Kido Y, Mori M, Sugimachi K. Comparison of characteristics of esophageal squamous cell carcinoma associated with head and neck cancer and those with gastric cancer. J Surg Oncol 1991;46:107-9. 2. Hanaoka T, Tsugane S, Ando N, Ishida K, Kakegawa T, Isono K, et al. Alcohol consumption and risk of esophageal cancer in Japan: a case-control study in seven hospitals. Jpn J Clin Oncol 1994;24:241-6. 3. Akiba S, Hirayama T. Cigarette smoking and cancer mortality risk in Japanese men and women—results from reanalysis of the six-prefecture cohort study data. Environ Health Perspect 1990;87:19-26. 4. Koga Y, Kuwano H, Sugimachi K. Biological characteristics of esophageal epithelial dysplasia assessed by proliferating cell nuclear antigen. Cancer 1996;77:237-44. 5. Kuwano H, Watanabe M, Sadanaga N, Ikebe M, Mori M, Sugimachi K. Squamous epithelial dysplasia associated with squamous cell carcinoma of the esophagus. Cancer Lett 1993;72:141-7. 6. Wagata T, Shibagaki I, Imamura M, Shimada Y, Toguchida J, Yandell DW, et al. Loss of 17p, mutation of the p53 gene, and overexpression of p53 protein in esophageal squamous cell carcinomas. Cancer Res 1993;53:846-50.
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7. Bennett WP, Hollstein MC, Metcalf RA, Welsh JA, He A, Zhu SM, et al. p53 mutation and protein accumulation during multistage human esophageal carcinogenesis. Cancer Res 1992;52:6092-7. 8. Gao H, Wang LD, Zhou Q, Hong JY, Huang TY, Yang CS. p53 tumor suppressor gene mutation in early esophageal precancerous lesions and carcinoma among high-risk populations in Henan, China. Cancer Res 1994;54:4342-6. 9. Wang LD, Hong JY, Qiu SL, Gao H, Yang CS. Accumulation of p53 protein in human esophageal precancerous lesions: a possible early biomarker for carcinogenesis. Cancer Res 1993;53:1783-7. 10. Morita M, Kuwano H, Ohno S, Sugimachi K, Seo Y, Tomoda H, et al. Multiple occurrence of carcinoma in the upper aerodigestive tract associated with esophageal cancer: reference to smoking, drinking and family history. Int J Cancer 1994;58:207-10. 11. Saeki H, Ohno S, Araki K, Egashira A, Kawaguchi H, Ikeda Y, et al. Alcohol consumption and cigarette smoking in relation to high frequency of p53 protein accumulation in oesophageal squamous cell carcinoma in the Japanese. Br J Cancer 2000;82:1892-4. 12. Greenblatt MS, Bennett WP, Hollstein M, Harris CC. Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res 1994;54:4855-78. 13. Harris CC. p53: at the crossroads of molecular carcinogenesis and risk assessment. Science 1980;262:1980-1. 14. Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers. Science 1991;253:49-53.
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15. Levine AJ, Momand J, Finlay CA. The p53 tumour suppressor gene. Nature 1991;351:453-6. 16. Rogel A, Popliker M, Webb CG, Oren M. p53 cellular tumor antigen: analysis of mRNA levels in normal adult tissues, embryos, and tumors. Mol Cell Biol 1985;5:2851-5. 17. Bennett WP, Hollstein MC, He A, Zhu SM, Resau JH, Trump BF, et al. Archival analysis of p53 genetic and protein alterations in Chinese esophageal cancer. Oncogene 1991;6: 1779-84. 18. Bodner SM, Minna JD, Jensen SM, D’Amico D, Carbone D, Mitsudomi T, et al. Expression of mutant p53 proteins in lung cancer correlates with the class of p53 gene mutation. Oncogene 1992;7:743-9. 19. Nakanishi Y, Nogichi M, Matuno Y, Saikawa M, Mukai K, Shimosato Y, et al. p53 expression in multicentric squamous cell carcinoma and surrounding squamous epithelium of the upper aerodigestive tract. Immunohistochemical analysis of 95 lesions. Cancer 1995;75:1657-62. 20. Yokoyama A, Muramatsu T, Ohmori T, Makuuchi H, Higuchi S, Matsushita S, et al. Multiple primary esophageal and concurrent upper aerodigestive tract cancer and the aldehyde dehydrogenase-2 genotype of Japanese alcoholics. Cancer 1996;77:1986-90. 21. Kuwano H, Ohno S, Matsuda H, Mori M, Sugimachi K. Serial histological evaluation of multiple primary squamous cell carcinoma of the esophagus. Cancer 1988;61:1635-8. 22. Wang X, Christiani D, Mark E, Nelson H, Wiencke J, Gunn L, et al. Carcinogen exposure, p53 alteration, and K-ras mutation in synchronous multiple primary lung carcinoma. Cancer 1999;85:1734-9.
Background. The unique pathologic features of esophageal tumors in patients with esophageal cancer includes the presence of multiple occurrence within the esophagus. The aim of this study is to clarify the molecular mechanism of carcinogenesis of multiple esophageal squamous cell carcinomas in the Japanese. Methods. We studied the relationship between the incidence of patients with multiple carcinomas and the coexistence of dysplasia lesions with p53 protein accumulation, alcohol consumption, and cigarette smoking. Among 76 cancer lesions and 60 cases of dysplasia, p53 accumulation was studied by means of immunohistochemical analysis. Results. The incidence of patients with multiple carcinomas in the high-risk group was 33%, and the incidence of patients with a coexistence of dysplasia in the high-risk group was 67%. The incidence of patients with multiple carcinomas or the coexistence of dysplasia in the high-risk group was much higher than that of the middle-risk and low-risk groups (P < .0001 and P = .04, respectively). The average number of abnormal epitheliums, such as cancer and dysplasia, in the high-risk group was 3.2 ± 2.1. The average number of abnormal epitheliums was much higher than that of the other groups (P = .02). For carcinoma lesions, the incidence of lesions with a positive p53 protein accumulation in the high-risk group was 91%. Regarding dysplasia lesions, the incidence of lesions with a positive p53 protein accumulation in the high-risk group was 80%. The incidence of both cancer and dysplasia lesions with a positive p53 protein accumulation in the high-risk group was higher than that of the other groups. Conclusions. The pattern of p53 accumulation in dysplasia in the high-risk group was closely similar to that in cancer of the high-risk group. These findings support the concept of field carciogenesis of the esophagus. (Surgery 2002;131:S7-13.) From the Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
IN PATIENTS WITH SQUAMOUS cell carcinoma of the esophagus, one of the most unique pathologic features of the tumors is the presence of multiple occurrence within the esophagus. We found the incidence of this multiplicity to be 25.6% in patients not given preoperative radiation treatment.1 It is important to study the carcinogenesis of multiple occurrences in esophageal squamous cell carcinoma. Epidemiologic studies revealed that alcohol consumption and cigarette smoking are the major risk factors of squamous cell carcinoma of the Reprint requests: Mitsuhiro Miyazaki, MD, Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 8128582, Japan. Copyright © 2002 by Mosby, Inc. 0039-6060/2002/$35.00 + 0 11/0/119288 doi:10.1067/msy.2002.119288
esophagus. In Japan the estimated world age-standardized annual incidence of esophageal cancer is 8.9/100,000 for men and 1.5/100,000 for women. Alcohol consumption and cigarette smoking are considered to be the major risk factors for squamous cell carcinoma of the esophagus in Japanese persons living in Japan. Alcohol consumption is associated with an increased risk of esophageal cancer, and the amount of alcohol consumed was found to be the main risk determinant.2 A statistically significant dose-response relationship was noted between cigarette smoking and the mortality rate for carcinomas of the esophagus in men.3 Some studies, such as immunohistochemical detection of proliferation cell nuclear antigen for resected specimens, demonstrated that the biologic nature of the dysplasia might well be as serious a lesion as cancer itself.4 Carcinomas coexistent with dysplasia have been reported to be more frequentSURGERY S7
S8 Miyazaki et al
Fig 1. Distribution of the patients in this study on the basis of drinking index and pack-years.
ly seen in less advanced esophageal cancer.5 As a result, dysplasia is considered to be a good subject to study the cancerization of squamous cell carcinoma of the esophagus. We studied dysplasias in the patients with squamous cell carcinoma of the esophagus limited to the submucosal layer. p53 is one of the well-known oncosuppressor genes related to the tumor carcinogenesis in esophageal squamous cell carcinoma.6 Immunohistochemical analyses with specific antibodies to p53 showed that an abnormal accumulation of p53 protein was recognized at precancerous stages, such as basal cell hyperplasia and dysplasia of the esophagus, suggesting that p53 mutations are indeed important events in the early stage of esophageal squamous cell cancerization.7-9 The expression of p53 protein in esophageal squamous cell carcinoma tissue and dysplasia tissue taken from Japanese patients was immunohistochemically investigated, and the relationship between the incidence of patients with multiple cancer lesions and dysplasia and p53 protein accumulation and the patient’s history of alcohol consumption and cigarette smoking was analyzed. The aim of our study was to elucidate the molecular mechanism involved in the occurrence of multiple esophageal dysplasia and squamous cell carcinoma. MATERIALS AND METHODS Patients. Our study was based on 68 patients with primary esophageal squamous cell carcinoma surgically treated in our institute from 1989 to 1998. The main tumors of all patients were limited to the submucosal layer. In addition, none of the
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patients had been given either preoperative irradiation or chemotherapy. Microscopic sections of the whole resected esophagus were prepared from step-sectioned blocks measuring 5 mm in width and were stained with hematoxylin and eosin. The differentiation of squamous cell carcinoma and the TNM stage were determined on the basis of histologic evaluations. Regarding the histologic diagnosis of squamous epithelial dysplasia, we used the criteria defined by the World Health Organization’s International Histological Classification of Tumors in 1990, according to which the nuclei of the lesion are enlarged and hyperchromatic while showing an increased mitotic activity. In the case of mild dysplasia, cells with such atypical nuclei were limited to the basal layer. With the increasing grade of dysplasia, there was a progressive increase of the cells with atypical nuclei until the entire thickness of the epithelium was replaced. Then moderate dysplasia was diagnosed when the zone with atypical cells expanded up to one half of the epithelium. Severe dysplasia was diagnosed when the zone with atypical cells expanded up to three quarters of the epithelium. In addition, intraepithelial carcinoma was diagnosed when an epithelium was replaced with atypical cells either completely or almost completely without any invasive growth. All sections with squamous cell carcinoma and dysplasia were selected for this study. The patients were retrospectively evaluated from the medical records of the Registry of Esophageal Cancer at the Second Department of Surgery, Kyushu University. To define the use of alcohol and cigarettes, we used the same questionnaire for all patients, as described previously.10,11 One drink of alcohol corresponded to 180 mL of sake (the most popular rice-wine alcoholic beverage in Japan), 120 mL of white liquor (shochu), 70 mL of whisky, and 720 mL of beer. A drinking index of the accumulated amount of ethanol was defined as the number of drinks per week times the number of years of drinking. For example, those with a history of drinking 360 mL of sake per day for 30 years had a score of 420 on the drinking index. In terms of the accumulated amount of smoking, “pack-years” was defined as follows: (Number of cigarettes per day/20) × Number of years of smoking.
For example, a patient with a history of smoking 20 cigarettes per day for 30 years was scored as at 30 pack-years. All patients were distributed on the basis of drinking index and pack-years, as shown in Fig 1. Immunohistochemistry. p53 immunohistochemical staining was performed on formalin-fixed,
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Fig 2. Incidence of patients with multiple cancer lesions or the coexistence of dysplasia for each group. The incidence of the patients with multiple cancer lesions was significantly higher than that of the other groups (P < .0001). The incidence of the patients with the coexistence of dysplasia was significantly higher than that of the other groups (P = .04).
paraffin-embedded esophageal tissue with squamous cell carcinoma or dysplasia from all patients. The sections were dewaxed in xylene and rehydrated with ethanol and then microwaved at 95°C in phosphate-buffered saline for 20 minutes. These sections were incubated with 0.3% hydrogen peroxidase in methanol to block endogenous peroxidase activity. Normal rabbit serum was applied for 20 minutes to reduce any nonspecific antibody binding. A mouse monoclonal antibody against p53 (DO-7; Dako, Glostrup, Denmark) with a 1:100 dilution was incubated overnight at 4°C. Staining was detected by using the streptavidin-biotin-peroxidase complex method. p53 protein accumulation was investigated in each specimen by investigators blinded to the risk-factor data. When immunoreactive neoplasia cells accounted for over 10%, a positive result was recorded. Statistical analysis. The data on the patient group were compared with either the chi-square test or the Student t test. The average number of dysplasias per one patient and p53 protein accumulation were compared in esophageal squamous cell carcinoma and dysplasia to evaluate the correlation between the risk-factor data and the incidence of the patients with multiple carcinomas or the coexistence of dysplasia. RESULTS Patient population. The clinicopathologic findings of all patients are listed in Table I. No significant difference was seen among the 3 groups (lowrisk, middle-risk, and high-risk groups) regarding sex, age, length, depth, location, and differentiation (data not shown). There were 76 carcinoma
lesions and 60 dysplasia lesions among the 68 patients. The average number of carcinoma lesions is 1.1 ± 0.4 per patient. The average number of dysplasia lesions was 0.8 ± 1.6 per patient. Relationship between the incidence of cases with multiple cancer or dysplasia and both alcohol consumption and cigarette smoking. Among the 68 patients with esophageal squamous cell carcinoma, 15 (22.1%) belonged to the high-risk group, 32 (47.0%) belonged to the middle-risk group, and 21 (30.9%) belonged to the low-risk group. Among the 68 patients with esophageal squamous cell carcinoma, 5 (7.4%) belonged to the group with multiple carcinomas, and 26 (38.2%) belonged to the group with a coexistence of dysplasia. All of the patients with multiple carcinomas belonged to the high-risk group. The incidence of the patients with multiple carcinomas in each group was 33%, 0%, and 0% in the high-risk, middle-risk, and low-risk groups, respectively. In addition, the incidence of patients with a coexistence of dysplasia in each group was 67%, 29%, and 32% in the high-risk, middle-risk, and low-risk groups, respectively. There was a significant correlation between the incidence of the patients with multiple carcinomas or the coexistence of dysplasia and the risk factors, as shown in Fig 2 (P < .0001, P = .04). Relationship between the average number of dysplasias per patient and both alcohol consumption and cigarette smoking. Among the 68 patients with esophageal squamous cell carcinoma, the average number of dysplasias per patient is 0.8 ± 1.6. In addition, the average number in each group was 1.7 ± 1.6, 0.8 ± 1.7, and 0.5 ± 1.0 in the highrisk, middle-risk, and low-risk groups, respectively,
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Fig 3. Average number of dysplasia lesions per patient with esophageal squamous cell carcinoma for each group. There is a significant difference between the average number of dysplasia lesions for the high-risk and low-risk groups (P = .01).
Table I. Clinicopathologic findings of all patients (n = 68) Sex (M/F) Age (y) Length (cm) Location Upper Middle Lower Depth Tis T1 Differentiation Well Moderate Poor Expression of multiple cancer Negative Positive Expression of dysplasia Negative Positive No. of carcinomas per patient No. of dysplasia lesions per patient Drinking index Pack-years
62/6 63.1 ± 9.3 (36-76) 3.2 ± 1.7 (1-10) 10 38 20 4 64 5 49 14 63 5 42 26 1.1 ± 0.4 (1-3) 0.8 ± 1.6 (0-8) 630 ± 634 (0-4190) 40 ± 36 (0-150)
Values in parentheses are ranges.
as shown in Fig 3. There was a significant difference regarding the average number of dysplasias per patient between the high-risk and low-risk groups (P = .01). Alcohol consumption, cigarette smoking, and p53 protein accumulation in esophageal squamous
cell carcinoma and dysplasia. p53 protein– immunoreactive cells are rare in the normal squamous epithelium. Among the 75 cancer lesions of 68 patients with esophageal squamous cell carcinoma, 46 (61%) belonged to the p53 protein accumulation–positive group and 30 (39%) to the negative group. Among the 60 dysplasia lesions of 68 patients, 38 (63%) belonged to the p53 protein accumulation–positive group and 22 (37%) belonged to the negative group. p53 protein immunoreactivity was observed in the nucleus of each cell. In most specimens in the p53 protein accumulation–positive group, the p53 protein immunoreactive neoplastic cells were diffuse throughout the carcinoma and dysplasia (Fig 4). Fig 5 shows the correlation between alcohol consumption, cigarette smoking, and p53 protein accumulation in both carcinoma and dysplasia. For the carcinoma lesions, the incidence of the lesions with positive p53 protein accumulation was 91%, 48%, and 45% for the high-risk, middle-risk, and low-risk groups, respectively. For dysplasia lesions, the incidence of lesions with positive p53 protein accumulation was 80%, 54%, and 45% for the highrisk, middle-risk, and low-risk groups, respectively. A significant correlation was seen between the incidence of the lesions with positive p53 protein accumulation and both alcohol consumption and cigarette smoking for carcinoma lesions (P = .001). For dysplasia lesions, no significant correlation was observed between the incidence of the lesions with a positive p53 protein accumulation and both alcohol consumption and cigarette smoking. The incidence of the lesions with positive p53 protein accu-
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Fig 4. Image of a cancerous lesion with a p53 protein–positive expression. In most specimens in the p53 protein accumulation–positive group, p53 protein–immunoreactive neoplastic cells were diffuse throughout the carcinoma and dysplasia, such as observed in this case.
Fig 5. Incidence of lesions with p53 protein accumulation in esophageal squamous cell carcinoma and dysplasia for each group. For the carcinoma, the incidence of lesions with p53 protein accumulation for the high-risk group was significantly higher than that of the other groups (P = .0014). For dysplasia, there is no significant difference among the 3 groups, but the incidence of the lesions with p53 accumulation tended to be higher than that of the other groups (P = .068).
mulation did tend to be higher in the high-risk group than in the other groups (P = .068). DISCUSSION p53 has come to the forefront of cancer research because it is commonly mutated in human malignant disease.12-15 The timing of p53 protein–related alterations in carcinogenesis and progression can vary with the tumor. In the case of the esophagus, p53 alterations can occur in the early stage of cancer and may thus confer a growth
advantage to preinvasive cells.7-9 In our previous study we reported that alcohol consumption and cigarette smoking were related to a high frequency of p53 protein accumulation in esophageal squamous cell carcinoma in the Japanese.11 As a result, in this study we used dysplasia of the patients with carcinoma limited to the submucosal layer that has been recognized as precancerous to investigate the relationship between such risk factors as alcohol drinking and cigarette smoking to an abnormality in p53 for carcinogenesis for multiple esophageal
S12 Miyazaki et al
cancer. We investigated the p53 protein expression in 68 cases of esophageal squamous cell carcinoma in Japanese patients by using a highly specific antibody to p53, and we correlated the results with the patients’ history of using alcohol and cigarettes, both important causative factors in esophageal squamous cell carcinoma. To our knowledge, this is the first report to focus on a correlation between the epidemiologic risk factors and the expression of a multiple occurrence of dysplasia and cancer of the esophagus and an aberrant expression of a tumor-suppressor gene. We previously reported that heavy smoking and alcohol consumption are clearly associated with multiple occurrences of upper aerodigestive tract cancer in Japanese patients.10 The multicentric occurrence of dysplasia and squamous cell carcinoma in the same esophagus was at a certain frequency.10 In this study the same results were also recognized. These findings suggest that the multiple occurrence of dysplasia and squamous cell carcinoma might be related to alcohol consumption and cigarette smoking. Because we especially used cases with early esophageal squamous cell carcinoma in this study, the results of this study reflect the relationship between the multiple occurrence of squamous cell carcinoma and such risk factors as alcohol consumption and cigarette smoking. Our findings show that the accumulation of p53 protein in both carcinoma and dysplasia is closely associated with large amounts of both alcohol and cigarette use. In our previous study the accumulation of p53 protein was also closely related to such risk factors as heavy alcohol consumption and cigarette smoking.11 These findings suggest that the p53 protein accumulation of dysplasia might be related to such risk factors as heavy alcohol consumption and cigarette smoking regarding squamous cell carcinomas of the esophagus. The incidence of the multiple occurrence of the dysplasia and p53 accumulation in the dysplasia was higher in the high-risk group, which was similar to the finding of squamous cell carcinomas of the esophagus. As a result, the multiple carcinogenesis of the squamous cell carcinoma of the esophagus might well be related to the p53 mutation of the squamous epithelium as a result of the carcinogens supplied by heavy alcohol consumption and cigarette smoking. In normal cells p53 is expressed at a low level and has a short half-life (6 minutes in the spleen),16 and a change in molecular configuration leads to a stabilization of the protein product so that its half-life is greatly extended. Immunohistochemical screen-
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ing for elevated protein levels followed by a sequence analysis represents an efficient strategy for the elevation of the p53 mutational spectrum.17,18 In the current study p53 protein accumulation was immunohistochemically screened in 68 patients with primary esophageal squamous cell carcinoma. A sequence analysis is needed in ongoing studies to elucidate the association between the risk factors and mutation of p53 in esophageal squamous cell carcinoma. Regarding “field carcinogenesis,” this term was used to represent various aspects. There is a viewpoint of synchronous or metachronous cancer development in multiple organs, such as upper aerodigestive tract cancer.1,19,20 The multiple occurrence of carcinoma within one organ suggests the possibility of field carcinogenesis.21,22 The term “field carcinogenesis” indicates a phenomenon of almost simultaneous multicellular carcinogenesis. On the basis of our findings, the main mechanism of the multiple occurrence of esophageal squamous cell carcinoma in patients with heavy alcohol consumption and cigarette smoking may be due to field carcinogenesis. Our findings show that the pattern of p53 accumulation of the dysplasia in each group was similar to that of cancer in each group. From these findings, we conclude that the multiple carcinogenesis of esophageal squamous cell carcinoma is related to a mutation of p53 in a large area in the same esophagus by the exposure of carcinogen caused by large amounts of alcohol drinking and cigarette smoking. REFERENCES 1. Kuwano H, Morita M, Tsutsui S, Kido Y, Mori M, Sugimachi K. Comparison of characteristics of esophageal squamous cell carcinoma associated with head and neck cancer and those with gastric cancer. J Surg Oncol 1991;46:107-9. 2. Hanaoka T, Tsugane S, Ando N, Ishida K, Kakegawa T, Isono K, et al. Alcohol consumption and risk of esophageal cancer in Japan: a case-control study in seven hospitals. Jpn J Clin Oncol 1994;24:241-6. 3. Akiba S, Hirayama T. Cigarette smoking and cancer mortality risk in Japanese men and women—results from reanalysis of the six-prefecture cohort study data. Environ Health Perspect 1990;87:19-26. 4. Koga Y, Kuwano H, Sugimachi K. Biological characteristics of esophageal epithelial dysplasia assessed by proliferating cell nuclear antigen. Cancer 1996;77:237-44. 5. Kuwano H, Watanabe M, Sadanaga N, Ikebe M, Mori M, Sugimachi K. Squamous epithelial dysplasia associated with squamous cell carcinoma of the esophagus. Cancer Lett 1993;72:141-7. 6. Wagata T, Shibagaki I, Imamura M, Shimada Y, Toguchida J, Yandell DW, et al. Loss of 17p, mutation of the p53 gene, and overexpression of p53 protein in esophageal squamous cell carcinomas. Cancer Res 1993;53:846-50.
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7. Bennett WP, Hollstein MC, Metcalf RA, Welsh JA, He A, Zhu SM, et al. p53 mutation and protein accumulation during multistage human esophageal carcinogenesis. Cancer Res 1992;52:6092-7. 8. Gao H, Wang LD, Zhou Q, Hong JY, Huang TY, Yang CS. p53 tumor suppressor gene mutation in early esophageal precancerous lesions and carcinoma among high-risk populations in Henan, China. Cancer Res 1994;54:4342-6. 9. Wang LD, Hong JY, Qiu SL, Gao H, Yang CS. Accumulation of p53 protein in human esophageal precancerous lesions: a possible early biomarker for carcinogenesis. Cancer Res 1993;53:1783-7. 10. Morita M, Kuwano H, Ohno S, Sugimachi K, Seo Y, Tomoda H, et al. Multiple occurrence of carcinoma in the upper aerodigestive tract associated with esophageal cancer: reference to smoking, drinking and family history. Int J Cancer 1994;58:207-10. 11. Saeki H, Ohno S, Araki K, Egashira A, Kawaguchi H, Ikeda Y, et al. Alcohol consumption and cigarette smoking in relation to high frequency of p53 protein accumulation in oesophageal squamous cell carcinoma in the Japanese. Br J Cancer 2000;82:1892-4. 12. Greenblatt MS, Bennett WP, Hollstein M, Harris CC. Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res 1994;54:4855-78. 13. Harris CC. p53: at the crossroads of molecular carcinogenesis and risk assessment. Science 1980;262:1980-1. 14. Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers. Science 1991;253:49-53.
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15. Levine AJ, Momand J, Finlay CA. The p53 tumour suppressor gene. Nature 1991;351:453-6. 16. Rogel A, Popliker M, Webb CG, Oren M. p53 cellular tumor antigen: analysis of mRNA levels in normal adult tissues, embryos, and tumors. Mol Cell Biol 1985;5:2851-5. 17. Bennett WP, Hollstein MC, He A, Zhu SM, Resau JH, Trump BF, et al. Archival analysis of p53 genetic and protein alterations in Chinese esophageal cancer. Oncogene 1991;6: 1779-84. 18. Bodner SM, Minna JD, Jensen SM, D’Amico D, Carbone D, Mitsudomi T, et al. Expression of mutant p53 proteins in lung cancer correlates with the class of p53 gene mutation. Oncogene 1992;7:743-9. 19. Nakanishi Y, Nogichi M, Matuno Y, Saikawa M, Mukai K, Shimosato Y, et al. p53 expression in multicentric squamous cell carcinoma and surrounding squamous epithelium of the upper aerodigestive tract. Immunohistochemical analysis of 95 lesions. Cancer 1995;75:1657-62. 20. Yokoyama A, Muramatsu T, Ohmori T, Makuuchi H, Higuchi S, Matsushita S, et al. Multiple primary esophageal and concurrent upper aerodigestive tract cancer and the aldehyde dehydrogenase-2 genotype of Japanese alcoholics. Cancer 1996;77:1986-90. 21. Kuwano H, Ohno S, Matsuda H, Mori M, Sugimachi K. Serial histological evaluation of multiple primary squamous cell carcinoma of the esophagus. Cancer 1988;61:1635-8. 22. Wang X, Christiani D, Mark E, Nelson H, Wiencke J, Gunn L, et al. Carcinogen exposure, p53 alteration, and K-ras mutation in synchronous multiple primary lung carcinoma. Cancer 1999;85:1734-9.