Japanese Journal of Clinical Oncology 32:327-331 (2002)
© 2002 Foundation for Promotion of Cancer Research
Low Incidence of p53 Mutations in Well-differentiated Tongue Squamous Cell Carcinoma in Japan
1 Central Laboratory and Radiation Biology, Aichi Cancer Center Research Institute, Nagoya, 2 Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Nagoya and 3 Department of Surgery and Surgical Basic Science, Kyoto University Graduate School of Medicine, Kyoto, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Background: Despite an increasing number of patients suffering from squamous cell carcinomas of the tongue, little is known about the molecular mechanisms involved in the origin and development of these neoplasms.
Methods: We screened microdissected tongue squamous cell carcinoma (TSC) specimens from 28 consecutive, previously untreated, Japanese patients for mutations in the p53 tumor-suppressor gene single-strand conformation polymorphism analysis (exons 5, 6, 7, 8) and direct genomic sequencing.
Results: Among them, 24 tumor specimens were well differentiated, three moderately and one poorly differentiated, according to the WHO classification. Mutations in the p53 tumor-suppressor gene were detected in only two out of the 28 (7%) tumor specimens. One was well differentiated and the other was poorly differentiated.
Conclusions: Our results suggest that p53 gene mutations are less frequent in well differentiated TSC. These results indicate that mutations in the p53 gene may not be strongly involved in the development of well differentiated TSC.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Squamous cell carcinoma (SCC) of the head and neck is the sixth most frequent cancer in the world (1). In Japan, the number of annual deaths from all oral cancers (lip, oral, oropharyngeal and salivary gland cancer) showed a 4.5-fold increase from 698 to 3155 from 1950 to 1993. Tongue cancer was the most common and contributed to about 40% to 60% of all oral cancer deaths (2). In histology, SCC is the most common malignancy in the oral cavity.
Tongue squamous cell carcinoma (TSC) is characterized by relatively poor prognosis and treatment is generally followed by severe dysfunction and disfigurement. In the search for effective prevention, diagnosis and treatment of this cancer, genetic changes involved in the origin of oral TSC should be determined (3). Although the tumor suppressor gene p53 is the most frequently mutated gene in a wide variety of human cancers, it is not clear whether the p53 gene is also involved in TSC since few studies have been reported previously on p53 mutation in TSC. For an accurate DNA analysis of human tumor tissue samples, it is important to eliminate the multiple types of cells other than tumor cells present in the specimens. In this study, we screened 28 microdissected TSC specimens for mutations of the p53 gene by single-strand conformation polymorphism analysis (SSCP) (exons 5, 6, 7, 8) and direct genomic sequencing.
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PATIENTS AND METHODS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tumor Samples
Tumor tissues were obtained from 28 consecutive, previously untreated, Japanese patients with TSC, who underwent biopsy at Nagoya University Hospital from 1992 to 1995. There were 23 males and five females and their mean age was 57 years, ranging from 35 to 76 years. All TSC was detected from lateral tongue specimens taken before any treatment. The histological diagnosis of SCC was reconfirmed from hematoxylin–eosin stained sections. There were 24 well, three moderately and one poorly differentiated carcinomas, according to the WHO classification. The tumor staging (TNM) was determined according to the 1987 classification of UICC (4). There were seven, six, six and nine TSC patients in stages I, II, III and IV, respectively.
DNA Extraction
To extract DNA from tumor tissues, a slightly modified, microdissection technique of Zhuang et al. (5) was used. Tumor samples were fixed in 10% buffered formalin and embedded in paraffin for conventional histological examination and a 5 µm thick section was stained with hematoxylin and eosin. The DNA was extracted from the adjacent 5 µm thick sections. Unstained 5 µm thick tissue sections, on glass slides, were deparaffinized with xylene, rinsed with 95% ethanol, lightly stained with eosin and air-dried. Under a light microscope, specific histological areas of target cell populations were selected and scraped off.
Scraped 3–5 mm2 tissue was placed in a 1.5 ml microcentrifuge tube and washed with xylene and ethanol. The specimens were dried under vacuum and the resultant tissue fragments were gently digested with proteinase K (0.3 µg/ml) in 20 µl of Na-free TE buffer (1 mM EDTA, 10 mM Tris–HCl, pH 7.5) at 37°C overnight. The specimens were heated at 100°C for 5 min to inactivate proteinase K and the DNA solution was quantitated spectrophotometrically at 260 nm.
PCR–SSCP Analysis
PCR was performed in 50 µl of reaction mixture containing 100–150 ng of genomic DNA, 20 pmol of each primer, 200 µM of dATP, dGTP and dTTP, 20 µM of dCTP, 0.1 µCi of [32P]dCTP (3000 Ci/nmol) and 0.5 unit of AmpliTaq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT). We used five pairs of primers (Table 1) to amplify exons 5–8 and their flanking intron sequences in 30 reaction cycles (6). Each cycle consisted of 1.3 min of denaturation at 94°C, 1.5 min of annealing at 60°C and 2 min of polymerization at 72°C. For SSCP analysis (7), 5 µl aliquots of PCR product were diluted with 5–20 µl of a 0.1% SDS and a 10 mM EDTA mixture and then mixed with a loading solution (95% formamide, 89 mM Tris, 2 mM EDTA, 89 mM boric acid, 0.05% bromophenol blue, 0.05% xylene cyanol) at 1:1. Diluted samples were heat-denatured at 99°C for 5 min and then loaded on a 6% neutral polyacrylamide gel with or without 10% glycerol. After electrophoresis, gels were dried and exposed to X-ray film or an imaging plate (FUJIFILM, Tokyo, Japan). Samples with shifted bands were further analyzed by direct DNA sequencing.
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The DNA from esophageal cancer cell lines with known mutations (14) was used as a positive control for point mutations in each exon. A normal tissue without any p53 mutation was used as a negative control.
Direct DNA Sequencing
PCR amplification was carried out under the same conditions as above, except that 200 mM dCTP was added instead of [32P]dCTP. After the amplification, primers and nucleotides were removed from the reaction mixture with Ultrafree C3TK (Takara, Tokyo, Japan) and purified PCR products were directly sequenced with a Sequencing PRO (Toyobo, Osaka, Japan). For each sequencing reaction, one of two primers used for PCR was end-labeled with 32P using a Megalabel kit (Takara, Tokyo, Japan) and was used as a sequencing primer.
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RESULTS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DNAs from 28 tumors were screened for p53 mutations in exons 5, 6, 7 and 8 by PCR–SSCP analysis. Two of the 28 tumors (7%) exhibited extra bands in the SSCP analysis of exon 5 or exon 6 (Figs 1 and 2), indicating mutations. Direct sequencing revealed that they were single-base substitutions. These samples exhibit both mutant band and normal band in sequencing ladders and seem to contain contamination of stroma cells with normal p53. Tumor No.7 had mis-sense mutation in codons 203 (GTG
GAG) and tumor No.28 had non-sense mutation in codons 146 (TGGTGA) (Table 2). Although there have been a few previous studies on the p53 mutations in TSC, the mis-sense mutation in codons 203 has not been reported, to our knowledge. The non-sense mutation in codons 146 has been reported (8).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One of the most exciting aspects of human cancer research is the genetic alterations that occur in neoplasia. However, for studies of the genetic changes in premalignant and more advanced infiltrating tumors, one of the limiting factors that precludes an accurate DNA analysis of human tissue samples is the multiple cell types present in the specimens (5). For example, genetic analysis of homogenized tumor samples reflects the average content of all cell types present in the sample including normal epithelial cells, stromal cells, endothelial cells and inflammatory cells. Thus, tumor cell-specific alterations may not be discernible by molecular analysis. Histological sections of tumor specimens can be microdissected and small, specific regions of interest can be analyzed.
Reported incidences of p53 mutations involved in TSC vary from 12.5 to 66.7%: 12.5% (1/8) in India (9); 42.9% (3/7) in Japan (10); 54% (21/39) in Finland (11); and 66.7% (4/6) in France (12). The much lower mutation frequency of p53 detected in the present study is unlikely to be a result of the low detection power of our analysis since the same screening protocol was able to detect frequent p53 mutations in other kinds of tumor samples (6,13–15) and we analyzed exons 5–8, where 95% of the reported p53 mutations are localized (16). Most p53 mutations in the head and neck SCC were also found in the region between exons 5 and 8 (1,3). Hence, although possible, it is unlikely that we missed a few mutations in the SSCP analysis and also other mutations in other exons.
Of the two mutations observed in the present study, only one was detected in our 24 well differentiated TSC samples, suggesting that p53 mutations in the well differentiated TSC are not frequent. From the few studies on the relationship between p53 gene mutation and histological grading of TSC (8,17,18), only one of 14 p53 mutations was in well differentiated TSC (7%), seven in moderately differentiated TSC (50%) and six in poorly differentiated TSC (43%). Therefore, the frequency of p53 mutation in well differentiated TSC is relatively low. Atula et al. (11) also found that altered p53 SSCP pattern (mutation) was more frequent in the high-grade TSC than in the low-grade TSC. Nishioka et al. (19) and Shintani et al. (20) reported on p53 protein that the incidence of p53-overexpressed tumors was higher in poorly differentiated tumors. Furthermore, Ranasinghe et al. (21) noted that the majority of oral SCC not showing p53 overexpression were well differentiated tumors.
It is speculated that the tumor tissues in which there were no mutations of the p53 gene had other genetic alterations which may be either equivalent to inactivation of the normal function of the p53 protein or involved in other carcinogenic pathways. For example, the product of the MDM2 gene is known to bind to p53 protein and inhibit its ability to activate transcription. In some tumors such as sarcomas, amplification of the MDM2 gene plays an important role in tumorigenesis, causing loss of the normal function of the p53 gene (22). The E6 proteins of HPV types16 and 18 are considered to have transforming ability by binding to the p53 protein and inhibiting its function through a ubiquitin-dependent proteolysis system. Indeed, HPV infection is closely associated with the development of female genital epithelial cancers and over 90% of cervical cancer biopsy specimens contain high-risk HPV such as types of 16 and 18 (23). In head and neck SCC, one study found HPV-16 in 21% of tumors (24). Further investigation on HPV involvement and/or other genetic changes with a large number of cases is necessary to elucidate the molecular mechanisms responsible for the development of TSC, especially those in well differentiated ones.
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FOOTNOTES |
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+ For reprints and all correspondence: Shin-Ichi Tsurusako, Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: s-tsuru@med.nagoya-u.ac.jp
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Received February 21, 2002; accepted May 15, 2002
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