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p53 Tumor Suppressor Gene Mutation in Early Esophageal Precancerous Lesions and Carcinoma among High-Risk Populations in Henan,

China
Hongkun Gao, Li-Dong Wang, Qi Zhou, et al. Cancer Res 1994;54:4342-4346.

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[CANCER RESEARCH 54. 4342-4346,

August 15. 1"4|

p53 Tumor Suppressor Gene Mutation in Early Esophageal Precancerous Lesions and Carcinoma among High-Risk Populations in Henan, China1
Hongkun Gao, Li-Dong Wang, Qi Zhou, Jun-Yan Hong, Tong-Yuh Huang, and Chung S. Yang2
l.aborator\ for Cancer Research, College of I'liarmacv, Rutgers University, Piscataway, New Jersey 08855 H.G., L-D. W., Q. Z., T-Y. H., J-Y. H., C. S. Y.j, and Her-anInstitute of Medical Sciences, Henan Medical University, Zhengzhou 450052, China /L-D. W., Q. Z.

p53 protein in esophageal epithelia with dysplasia and hyperplasia and even in the papillae of near normal epithelia of biopsy samples from To understand whether /o.i gene mutation is an early or late event in individuals without cancer (17). The current study extended this line esophageal carcinogenesis, biopsy samples of esophageal epithelium from of investigation and analyzed p53 mutations in precancerous lesions symptom-free subjects in a high incidence area, Huixian county of Henan of the esophagus. Province, China, were analyzed. Mutations in exons 5, 6, 7, and 8 of p53 Mutations of the p53 tumor suppressor gene have been identified in were analyzed by single-strand conformation polymorphism analysis and many types of human cancers (18-21). More than 300 different point DNA sequencing. Among the 37 biopsy samples showing accumulation of mutations have been reported and several hot spots have been located p53 protein in immunohistochemical staining, missense mutations of p53 gene were delected in 1 of 3 samples with normal epithelia, 3 of 23 samples in the p53 gene, such as at codons 175, 248, and 273 (22). One hot with basal cell hyperplasia, and 4 of 11 samples with dysplasia. All spot of p53 mutation at codon 249 was detected in hepatocellular mutations occurred at exon 5 with 3 at codon 175, 2 at codon 176, and 1 carcinomas in patients from Qidong, China, and South Africa where each at codons 159, 135, and codon 132. Of the 8 mutations, there were 3 aflatoxin B, was a common risk factor (23, 24). In the same type of G to A transitions and 3 G to T transversions. To understand the mutation cancer from other areas where the populations were not exposed to spectrum and possible causative factors of esophageal cancer in this area, aflatoxin B,, mutations were detected at diverse positions of the p53 surgically resected human primary esophageal carcinomas from Linxian gene (25, 26). This suggests that the mutation pattern of p53 could be county were analyzed for p53 gene mutations in exons 5, 6, 7, and 8. related to the nature of the exposed environmental carcinogens (21). Mutations were detected in 16 of 29 samples (55%). Twelve samples contained different missense point mutations, with 75% transitions (7 G to It is possible that the high incidence of esophageal cancer in Henan, A and 2 A to G) and 25% transversions (2 G to T and l G to C). Most of China, is due to particular environmental carcinogens: and that mu the mutations were located at either exon 5 or exon 7. A deletion and an tation hot spots of the p53 gene exist in esophageal carcinomas and insertion of nucleotides leading to frame-shift mutations were detected in precancerous lesions. To examine this possibility, the mutation spec each of two other samples. The results demonstrate that p53 protein trum of the esophageal carcinomas was analyzed and compared with accumulation and gene mutation may occur at very early stages of esoph that of the precancerous lesions. ageal carcinogenesis. In carcinomas, there was a higher frequency ol'/o.f ABSTRACT
gene mutations, accumulation. which accounts for most of the cases with p53 protein

MATERIALS

AND METHODS
The biopsy samples were collected from symptom-

Specimen Collecting.

INTRODUCTION Esophugcal cancer is one of the most common cancers worldwide with sharp variation in its incidence rates among different countries, distinct geographic areas, and different ethnic groups (1). In Western countries, heavy cigarette smoking and alcohol intake are major risk factors, whereas in Third World countries, exposure to dietary car cinogens and nutritional deficiencies are believed to be major etiological factors (1-5). Linxian and the nearby counties in the Henan Province in northern China have received worldwide attention over the past decades because of their high esophageal cancer incidence (2). Ingestion of dietary carcinogens, in particular A'-nitroso com pounds and their precursors, has been implicated (2, 6, 7). Elevated level of O''-methyldeoxyguanosine in the esophageal cancer tissues has been reported (8). A'-Nitroso compounds induced esophageal tumors in rats and mice (9-11) and may be causative factors for human esophugeal cancer. However, the identities of the causative factors of human esophageal cancer still remain to be studied. Many pathological and epidemiological studies suggest that malig nant transformation of human esophageal mucosa is a progressive process (2, 12-16). Our previous studies revealed the accumulation of
Received 3/31/94; accepted 6/13/94. The costs of publication of this article were defrayed in part by Ihe payment of page charges. This article musi therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by NIH Grant CA37037 and National Institute of Envi ronmental Health Sciences Center Grant ES05022. whom requests for reprints should he addressed, To at Laboratory for Cancer

free subjects from Huixian, China. The surgically resected specimens were obtained from patients with primary esophugeal carcinoma in Linxian. China. The patients had not received any radiation therapy or chemotherapy before surgery. The pathological diagnosis was based on hematoxylin and eosin staining and all samples were invasive SCC.' Immunohistochemical Analysis of p53 Protein. Immunohislochemical analysis was performed using the avidin-biotin-peroxidase complex method as described previously ( 17). The primary antibody was a monoclonal mouse antibody raised against human p53 protein and obtained from Oncogene Science (Uniondale, NY). The avidin-biotin-peroxidase complex elite reagent and chromogen diaminoben/idine were from Vector Laboratories, Inc. (Burlingame. CA). Intense dark staining was the criterion for a positive reaction. Several terms were used to describe the immunostaining patterns: papillary, where immunoreactive cells were identified only in the papillary area; scat tered, in which only some isolated positive cells were identified; focal, where clusters of positive cells were seen in some areas of the epithelium: and diffuse, in which the sheets of positive cells were found throughout most areas of the lesions. In each experiment, positive and negative controls were included. The positive control was the esophageal carcinoma tissue which consistently showed positive staining in previous experiments. In the negative control, primary antibody was not added. The second negative control was the esoph ageal epithelium, which consistently showed negative staining in previous experiments. DNA Preparation. Tissues from surgically resected samples were cut into about 1-mm1 pieces with a scalpel, washed with H,O, and then incubated for 4 h at 55Cin a lysis solution (Applied Biosystems. Foster City, CA) plus proteinasc K and RNase A with the final concentration at 250 and l(X) fig/ml, respectively. Then the solution was successively extracted with equal volumes
3 The abbreviations used are: SCC. squamous cell carcinoma; BCH. basal cell hyper plasia; SSCP. single strand conformation polymorphism; PCR. polymerase chain reaction.

Research. College of Pharmacy. Rutgers University. Piscataway, NJ 08855. 4342

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of Tris-saturated

phenol (pH 8.0):chloroform:isoamyl

alcohol (25:24:1, v/v/v)

until the protein interface became invisible. DNA was precipitated with ethanol and DNA pellet was dried and dissolved in sterile water. The concentration of DNA was determined by spectrophotometry. With the use of biopsy samples and some resected tumor tissues, areas with p53 protein accumulation were first identified by immunostaining in one of the slides. Cells were then scraped from the corresponding areas in the unstained serial sections. The dissected cells were digested in a lysis solution containing 50 ITIM Tris (pH 8.0), l mM EDTA, 0.5% Tween 20, and 200 fig/ml proteinase K. The digestion mixture was used directly in PCR without further purification. Polymerase Chain Reaction. Oligonucleotide primers were synthesized by American Synthesis. Inc. (Pleasanton, CA). The recombinant Taq DNA polymerase was purchased from Perkin Elmer Cetus (Norwalk, CT). Deoxynucleotides were obtained from Pharmacia (Piscataway. NJ). The PTC-HK) Programmable Thermal Controller was obtained from MJ Research, Inc. Four fragments of DNA, including exons 5, 6, 7, and 8 of the p53 gene, were amplified. The following primers were used: 5'-GTT TCT TTG CTG CCG TGT TC-3'. 5'-AGG CCT GGG GAC CCT GGG CA-3' for exon 5; 5'-TGG TTG CCC AGG GTC CCC AG-3', 5'-AGG AGO GCC ACT GAC AAC CA-3' for exon 6; 5'-ACC ATC CTG GCT AAC GOT GA-3', 5'-AGG GGT CAG CGG CAA GCA G-3' for exon 7; and 5'-TTG GO A GTA GAT GGA GCC CT-3'. 5'-AGO CAT AAC TGC ACC CTT GG-3' for exon 8. PCR was carried out in 100 /xl of reaction containing 50 mM Tris (pH 9.0), 30 mM MgCK, 100-500 fig of genomic DNA. 40 pmol of each primer, 2(H) JIM concentrations of each of the four deoxynucleotide triphosphates. and 5 units of Taq polymerase, topped with 50 jxl of light mineral oil. Thermal cycles consisted of an initial 5 min at 95Cbefore the addition of Taq polymerase, followed by 35 cycles of 30 s at 94C,1 min at 60C,and 30 s at 76C.PCR products were purified from 1.5% agarose gel using the Mermaid kit from Bio 101 (La Jolla, CA). SSCP Analysis. PCR products of exons 5, 6, 7, and 8 of the p53 gene were labeled at the 5' ends with [7-'2P]ATP. No purification of the labeled products was required. Five % of the reaction solution was run on ft% polyacrylamide gel (29:1 and 99:1 acrylamide:bisacrylamide). For exons 6 and 8. three additional gel conditions were used: MDE gel (AT Biochem, Malvern, PA); 6% polyacrylamide (99:1); and 12% polyacrylamide (29:1) with 10% glycerol. The gel was run at 30 W for 8-12 h at room temperature and cooled with a fan. Human placental DNA (Oncogene Science) was used as normal control. The gel was then dried and exposed to X-ray film (Kodak) overnight. Any bands with shifted mobilities compared to normal control were cut out from the gel and eluted in H,O at 80Cfor 10 min. Five jil of this clute were amplified in PCR with 20 thermal cycles using the same pair of primers and the same conditions as used in the previous PCR. The PCR product was purified and checked by SSCP analysis to verify that the correct bands were amplified. When a shifted band in SSCP was detected, the experiment was always repeated to confirm the result. DNA Sequencing. Purified amplified DNA was directly sequenced by dideoxy chain termination method. The sequences of primers were nested to the PCR primers. Four pairs of primers were used to sequence both strands of DNA. Sequencing kit was from United States Biochemical Corp. (Cleveland. OH) and procedures were modified from their standard protocol. G>T

12345
Fig. 1. Representative SSCP analysis of p53 gene exon 5 in esophugeal squamous carcinomas and precancerous lesions. Lanes I and 4, negative controls; Lanes 2 and 3, normal adjacent tissue and tumor of sample A910487; Lane 5, biopsy sample AB202 with dysplasia. Arrows, hands showing mobility changes.

A910623 tumor

A910623 normal

GATC

GATC
5'

RESULTS p53 Gene Mutation and Protein Accumulation in Esophageal Carcinoma. In order to establish the relationship between p53 gene mutation and p53 protein accumulation, esophageal carcinomas were analyzed for mutations in exons 5, 6, 7, and 8 of the p53 gene. Representative examples of SSCP and DNA sequencing analyses are shown in Figs. 1 and 2. Eighteen of the 29 tumor samples (62%) showed accumulation of p53 protein in the nuclei by immunohistochemical staining. Most of the samples had a diffuse staining pattern (Table 1). Among these 18 staining positive samples, 12 had point mutations including 7 in exon 5, 4 in exon 7, and 1 in exon 8. In one sample, in addition to a point mutation in exon 5, there was also a 6-base pair insertion in the same exon (Table 1). Among these point mutations, 9 were transitions (7 G

Fig. 2. DNA sequence analysis of p53 gene exon 5 in esophageal squamous carcinoma A910623 and its adjacent normal tissue. Arrow, nucleotide substitution in codon 158 (CGC to CTC) in tumor.

to A and 2 A to G) and 3 were transversions (2 G to T and l G to C). DNA sequence analysis of the 11 /?5J-staining negative samples revealed 4 samples with/;5.? gene mutations. There were 2 frame-shift mutations by either a 5-base pair insertion at exon 7 or an 8-base pair deletion at exon 5. These two frame-shift mutations could produce truncated p53 proteins due to premature termination of the translation. There were also 1 silent mutation at codon 136 of exon 5 and 1 point mutation at intron 5. p53 mutations were not detected in the appar ently normal epithelia adjacent to the SCC in the resected esophagi in

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Table 1 p53 gene mutation and protein accumulation in esophageal carcinomas Samples were from male and female patients, 42-61 years old.

over cancer tissues; in the latter, molecular events may occur as a consequence of the neoplasm. The present study demonstrates that p53 protein accumulation occurs very frequently in dysplasia, a well mutationBase/amino of p53 recognized lesion of esophageal cancer (2, 12, 16). Accumulation of changesCGC acid immunostainingDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseFocalScatteredScatteredaaaFocalDiffuseaExon/codon5/1585/1595/1465/5/1737/2377/2397/2458/2805/1755/1755/1357 Subject9106239 p53 protein also occurs frequently in basal cell hyperplasia, which is LeuGCC .CTC/Arg ACC/Ala-.ThrTGG->TAG/Trp-->stop6-base considered 104979106749165391682910468910646924114924115911524910514910472917019107439115169104879104735 as a precancerous lesion by some investigators but not by others because of its reversibility. It is generally agreed, however, that insertionGTG pair basal cell proliferation predisposes the tissue to carcinogenesis (13>CTG/Val-LeuATO
-ATA/Met-IleAAC
>AGC/Ser-PheGGC GinAGA -GAC/Arg -GysCGC^CAC/Arg 'AAA/Arg

-HisCGC -HisTGC -CAC/Arg


-PheAAC .TTC/Cys -AspCAA-.CAG115-base -GAC/Asn frame-shift8-base pair insertion, 55/ecep53 pair deletion, frame-shift subjects7 subjectsPatlcrn " Immunostain negative. h No amino acid change. ' Mutation not detected in exons 5, 6, 7, and 8.

16). p53 protein accumulation appears to occur among these prolif erating cells (17). In preliminary studies, we found that the number of p53 immunostain-positive cells increased markedly from BCH to dysplasia and increased greatly from dysplasia to SCC.5 It is possible that p53 protein accumulation is an early event in carcinogenesis and that the proliferating cells positively stained with the p53 antibody in the papillae are the initiating sites of esophageal carcinogenesis, especially if p53 gene mutation occurs in these cells. The observation that p53 gene mutation was observed in normal epithelium suggests that such mutation may occur even before morphological changes can be observed. Among the p53-staining positive biopsy samples, 4 of 11 samples with dysplasia had point mutation in exon 5 of the p53 gene, whereas only 3 had mutations among 23 samples with BCH. It is possible that our present method was not sensitive enough to detect certain muta tions when the p53-staining positive cells were in low abundance. An

all the 29 cases in Table I,4 suggesting the mutations observed in SCC

were somatic events. attempt is being made to improve our scraping method to enrich the In order to exclude the possibility that lack of detected mutations at positively stained cells for mutation analysis. Another possibility is exon 6 ofp53 gene was caused by technical bias in our study, different that p53 mutation is not the molecular basis for the p53 protein gel compositions and running conditions were used in SSCP analysis accumulation or is an event subsequent to the protein accumulation in of exon 6 with a PCR fragment of exon 6 containing a point mutation some of the cases. These possibilities remain to be examined. as positive control. There was no mobility shift detected for the The results in the current study showed a good correlation between samples under these conditions. Direct sequencing of PCR products of the nuclear p53 protein immunostain and p53 gene mutations in 12 exon 6 demonstrated the normal sequence in all these samples. SCC samples, suggesting that the accumulated p53 proteins in the pS3 Gene Mutation and Protein Accumulation in Esophageal cancer cells are mostly the mutated forms. There were 6 cases, Precancerous Lesions. A total of 220 biopsy specimens of esopha however, in which p53 protein levels were high but p53 mutations geal epithelia were obtained from symptom-free subjects. Most of were not detected. In these 6 cases, it is possible that the accumulation them (90%) were immunostaining positive for p53 protein. Different of p53 protein was due to a p53 mutation-independent mechanism or staining patterns were observed and were classified as papillary, that the p53 mutations did occur but were not in exons 5, 6, 7, and 8. scattered, focal, and diffuse. The results were in accordance to our The observed mutations are most likely somatic events because mu previous study (17). The percentage of positive staining cells ranged tations were not observed in the 29 samples of noncancerous epithe from less than 5% (as in AB214) to 75% (as in AB202) of the total lium adjacent to the SCC in the resected esophagi. Germ line muta cell population. Generally, a higher percentage of p53-staining posi tions and polymorphisms with silent mutations have not been reported tive cells and a diffuse pattern were more frequently observed in samples with dysplasia than those with lower grade lesions. Thirty- for this population and remain to be investigated. Several common features have been observed between the p53 seven mmunostaining positive samples, including 3 normal epithelia, mutation spectra detected in this and other studies (18, 27-30). The 23 BCH, and 11 dysplasia, were analyzed for p53 gene mutation (Table 2). Point mutations were detected in 4 of the 11 dysplasia previously reported frequency of p53 mutations in human esophageal samples, 3 of the 23 BCH samples, and 1 of the 3 normal samples. All cancer is about 50% and G to A transitions are the predominant point of these point mutations were in exon 5, with 3 at codon 175; 2 at mutations (40% of total mutations). Similar frequencies of p53 mu codon 176; and 1 each at codons 159, 135, and 132. There were 4 tations (52%) and G to A transitions (42%) were seen herein. The G to A and A to G transitions may be a result of carcinogen-induced transitions (3 G to A and 1 A to G) and 4 transversions (3 G to T and alkylation of DNA. The formation of Oh-methylguanine may cause a l G to C). It was noted that when the percentage of the positively stained cells decreased, fewer mutations were detected among the mispair for thymine in DNA replication. In the subsequent round of samples. For example, mutations were detected in 4 of 10 samples DNA replication, an adenine would replace the original guanine and which contained 30-50% p53-staining positive cells but in only 2 of result in a G to A transition (31). On the other hand, the formation of O4-methylthymine would result in an A to G transition (10, 32). These 22 samples which contained less than 30% positively stained cells. mechanisms appear to be consistent with the hypothesis that nitrosamines are the carcinogens for human esophageal carcinogenesis in DISCUSSION Henan, China (2, 5). Alternatively, deamination of 5-methylcytosine by nitric oxide, which may be formed in esophagitis, would give rise For the study of the primary or early molecular events of carcinoto thymine (33). If this occurs at the noncoding DNA strand, the genesis, biopsy samples with precancerous lesions have the advantage coding DNA strand would have a G to A transition. The deamination
4 In subject 910497, the adjacent epithelium had dysplasia and a 3-base pair deletion at exon 6 of the p53 gene but no point mutation. 4344 ' Unpublished results.

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Table 2 p53 gene mutation and protein accumulation in biopsy samples from symptom-freesubjects

mutationExon/codon5/1765/1755/1595/1325/1765/1755/1355/175Base/amino of p53 immune-stainingDiffuseDiffuseDiffuseDiffuseDiffusePapillary, SubjectAB202AB218C-lC-23AB226AB203AB215AB214C-6AB2283 (yr)6338686534502753494021-5948-5047,6632,4443,4622-5731^571,60HistopathologyDysDysDysDysBCHBCHBCHNormalDysBCHDysDysBCHBCHBCHBCHB changeTOC acid ^TTT/Cys->PheCGC->CAC/Arg->HisGCC^CCC

*CTC/Arg->LeuTOC scatteredPapillaryPapillaryPapillaryFocalScatteredDiffusePapillary, ^TAC/Cys^TyrCGC-*CAC/Arg-^His a--subjects3 subjects2 subjects2 subjects2 subjects6 subjects7 subjects2 subjectsAge ' ,mutation not detected in exons 5, 6, 7, and 8.

diffusePapillary, scatteredPapillary, focalDiffusePapillaryPapillaryp53

of 5-methylcytosine

occurs predominantly

at the CpG site; in the

present study two G to A mutations occurred at CpG sites. In other studies on p53 mutation in human esophageal cancers, exons 5, 6, 7, and 8 were affected by mutations with similar frequen cies (18, 27-30). A major difference in our study is that most of mutations detected herein were in exons 5 and 7, with only 1 in exon 8 and none in exon 6. This difference in mutation profile may reflect the involvement of different causative agents in the esophageal carcinogenesis in Henan, China. Interestingly, all of the 8p53 mutations identified in biopsy samples from symptom-free subjects were located in exon 5 and 5 were at codons 175 and 176. This result suggests a nonrandom p53 mutation pattern during the early stage of esophageal tumorigenesis. This pattern was not observed in the esophageal SCC, in which p53 mutations were more diversely distributed and only 2 of 12 mutations were at codon 175. One possibility is that codons 175 and 176 of the p53 gene are within the most susceptible DNA sequences to the mutagenesis by particular carcinogens. The mutations at these two codons may render the cells some growth advantage but are not the only requirements for neoplastic transformation. The mutations de tected in esophageal SCC may have a stronger potential to cause neoplastic transformation by itself or in cooperation with other oncogenic products. The present results suggest that p53 gene mutation is an early event in esophageal carcinogenesis. This concept needs to be further substantiated in subsequent follow-up studies. ACKNOWLEDGMENTS
We are grateful to Drs. Curtis C. Harris and William P. Bennet and to J. A. Welsh of the National Cancer Institute, for technical advice in the initial phase of this work.

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