Role of p53 gene mutations in human esophageal carcinogenesis: results from immunohistochemical and mutation analyses of carcinomas and nearby non-cancerous lesions
Stephanie T. Shi,
Guang-Yu Yang,
Li-Dong Wang1,
Zhihong Xue,
Bo Feng,
Wei Ding,
Eric Poe Xing and
Chung S. Yang2
Laboratory for Cancer Research, College of Pharmacy, Rutgers University, Piscataway, NJ 088550789, USA and
1 Henan Medical University, Zhengzhou, Henan, China
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Abstract
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In order to characterize p53 alterations in esophageal cancer and to study their roles in carcinogenesis, we performed gene mutation and immunohistochemical analysis on 43 surgically resected human esophageal specimens, which contain squamous cell carcinoma (SCC) and adjacent non-cancerous lesions, from a high-incidence area of Linzhou in Henan, China. A newly developed immunohisto-selective sequencing (IHSS) method was used to enrich the p53 immunostain-positive cells for mutation analysis. p53 gene mutations were detected in 30 out of 43 (70%) SCC cases. Among 29 SCC cases that were stained positive for p53 protein, 25 (86%) were found to contain p53 mutations. In five cases of SCC with homogeneous p53 staining, the same mutation was observed in samples taken from four different positions of each tumor. In a well differentiated cancer nest, p53 mutation was detected in only the peripheral p53-positive cells. In tumor areas with heterogeneous p53 staining, either the area stained positive for p53 had an additional mutation to the negatively stained area or both areas lacked any detectable p53 mutation. In the p53-positive non-cancerous lesions adjacent to cancer, p53 mutations were detected in seven out of 16 (47%) samples with basal cell hyperplasia (BCH), eight out of 12 (67%) samples with dysplasia (DYS), and six out of seven (86%) samples with carcinoma in situ (CIS). All mutations found in lesions with DYS and CIS were the same as those in the nearby SCC. In seven cases of BCH containing mutations, only three had the same mutations as the nearby SCC. The results suggest that p53 mutation is an early event in esophageal carcinogenesis occurring in most of the DYS and CIS lesions, and cells with such mutations will progress to carcinoma, whereas the role of p53 mutations in BCH is less clear.
Abbreviations: BCH, basal cell hyperplasia; CIS, carcinoma in situ; DYS, dysplasia; IHSS, immunohisto-selective sequencing; SCC, squamous cell carcinoma; SSCP, single strand conformation polymorphism; wt, wild-type.
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Introduction
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Human esophageal carcinogenesis is a multistage progressive process, which involves the conversion of normal epithelium to that with basal cell hyperplasia (BCH), dysplasia (DYS) or carcinoma in situ (CIS), and then to invasive squamous cell carcinoma (SCC) (14). Immunohistochemical studies and mutation analysis in our and other laboratories have demonstrated that p53 protein accumulation and p53 gene mutation occur at early stages of esophageal carcinogenesis, including even mild BCH and near-normal epithelia (514). Due to technical limitations, early molecular changes in precancerous lesions have not been extensively studied. Recently, we developed an immunohisto-selective sequencing (IHSS) method (15) which enabled us to perform a more accurate p53 gene mutation analysis in p53 immunostain-positive precancerous lesions. With the IHSS method, we analysed p53 gene mutation in 43 surgically resected SCC specimens and the nearby p53 immunostain-positive non-cancerous lesions. Since the SCC and the adjacent non-cancerous lesions were from the same subjects, we were able to compare p53 mutations at different stages of human esophageal carcinogenesis. The relationship between p53 protein immunostaining and gene mutation was also analysed.
Intra-tumor heterogeneity in the distribution of the mutant p53 allele has been reported in human prostate cancer (16). In another study, however, a particular p53 gene mutation was identical in all tumor areas per case investigated in one case of esophageal cancer, five cases of stomach cancers, and 14 cases of colorectal cancers (17). It is important to understand intra-tumor heterogeneity because the outcome of mutation analysis would depend not only on which tumors, but also on which regions of the tumors are sampled in the study. It may also have important implications in the understanding of the origin of cancer cells and the timing of p53 gene mutation during esophageal carcinogenesis. To address this question, we have taken tissue samples at four distant sites from each primary tumor of the esophagus with homogeneous p53 immunostaining, and compared the results of p53 mutation analysis. In the well differentiated SCC nest, intense p53 staining was observed in the peripheral, but not in the interior, of the cancer nest. In other specimens, areas with heterogeneous p53 immunostaining within the primary tumor were observed. In one case, part of the tumor was stained positive and the other part was negative. In the other two cases, only a few cells were stained positive for p53 protein in an otherwise p53 immunostain-negative cancer field. We performed mutation analysis on all of these heterogeneously stained areas and compared the results in order to understand the molecular basis of the different staining patterns.
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Materials and methods
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Esophageal tissue specimens
Human esophageal tissue specimens were obtained at the time of surgery from patients with esophageal cancer from Linzhou (formerly Linxian) of the Henan Province in northern China, and fixed in 80% ethanol. The 43 patients were 3764 years old (mean 53.1) and evenly distributed in terms of sex. Parts of the specimens were embedded in paraffin for H & E staining and p53 immunostaining. All of the tissue blocks were cut into 5 µm sections and adhered to plastic transparencies (Xerox, Rochester, NY) that were cut to the size of the microscope slides. The tissue sections were then heated at 60°C in a hybridization oven for 30 min and cooled to room temperature before immunostaining.
Immunohistochemical analysis
The Vectastain Elite ABC kit and the DAB Substrate kit (Vector Laboratories, Burlingame, CA) were used for immunostaining as described previously (15). p53 Antibody, Ab-6, from Oncogene Science (Uniondale, NY) was used as the primary antibody. It should be noted that we used twice as much of the nickel solution as that suggested by the manufacturer to increase the darkness of the immunostaining. Each step was followed and timed exactly to ensure minimal and uniform background staining for all of the tissue sections.
DNA preparation from cancerous tissues
Tissue sections on plastic slides were immunostained with p53 antibody. The positive areas were dissected out to ensure at least 80% p53 immunostain-positive cells in the preparation. With p53 immunostain-negative SCC, the cancerous areas were dissected, based on H & E staining. They were then cut into small pieces and placed into Eppendorf tubes. The pieces were covered with 100200 µl of digestion buffer (100 mM TrisHCl, 2 mM EDTA, pH 8.0, 0.4 mg/ml proteinase K) and incubated at 55°C for 3 h.
In order to study intra-tumor heterogeneity of p53 gene mutation, tissues were taken at 3, 6, 9 and 12 o'clock positions from a single primary tumor for each specimen.
DNA preparation from non-cancerous lesions
For lesions with BCH, DYS and CIS, and some of the tumor sections with a low percentage of p53 immunostain-positive cells (<30%), UV irradiation was used to enrich the p53 immunostain-positive cells as described previously (15). In brief, the p53-immunostained slides were placed tissue side down on a UV trans-illuminator for UV irradiation. The length of time needed for the complete inactivation of DNA for PCR amplification was determined by irradiating sections that were stained negative for p53 protein for different lengths of time. Based on these results, esophageal specimens with different lesions were irradiated by UV for the desired lengths of time, i.e. 3, 4, 4.5 and 1.5 h for exons 5, 6, 7 and 8, respectively. Afterwards, the sections containing the p53 immunostain-positive cells were cut into small pieces with sterile scissors and placed into Eppendorf tubes. The pieces were covered with 2550 µl of digestion buffer and incubated at 55°C for 3 h.
Polymerase chain reaction
The primers used for PCR amplification of the p53 gene were 5'-ACT TCC TGA AAA CAA CGT TC-3' and 5'-CAG GCA TTG AAG TCT CAT GG-3' for exon 4, 5'-GTT TCT TTG CTG CCG TGT TC-3' and 5'-AGG CCT GGG GAC CCT GGG CA-3' for exon 5, 5'-TGG TTG CCC AGG GTC CCC AG-3' and 5'-GGA GGG CCA CTG ACA ACC A-3' for exon 6, 5'-AGG CGC ACT GGC CTC ATC TT-3' and 5'-AGG GGT CAG CGG CAA GCA GA-3' for exon 7, 5'-TTG GGA GTA GAT GGA GCC T-3' and 5'-AGG CAT AAC TGC ACC CTT GG- 3' for exon 8, and 5'-GCA GTT ATG CCT CAG ATT CA-3' and 5'-GGC ATT TTG AGT GTT AGA CT-3' for exon 9. The PCR primers amplified the splicing donor and acceptor sites for all exons examined. PCR was carried out in 20 µl of reaction buffer (50 mM Tris, pH 9.0, 30 mM MgCl2) containing 8 pmol of each primer, 200 µM of each dNTP, 1 µCi of [
-32P]dATP, and 0.5 U of Taq polymerase. The thermal cycles consisted of an initial cycle of 3 min at 95°C before the addition of Taq polymerase, followed by 40 cycles of 1 min at 94°C, 1 min at 60°C and 1 min at 72°C, and concluded by 10 min at 72°C for elongation. To avoid PCR contamination, a blank control without DNA was always included in every PCR reaction.
Single strand conformation polymorphism (SSCP) analysis
The labeled PCR products were mixed with 2 vol of TBE buffer containing 25 mM methylmercury hydroxide and loaded on a 6% polyacrylamide gel containing 5% glycerol. The gel was run at 8, 15 or 30 W at room temperature cooled with a fan. Human placental DNA was used as a negative control in SSCP and the subsequent DNA sequencing. Bands with different mobilities as compared with the placental control were eluted and amplified by a second PCR. The thermal cycles consisted of 35 cycles of 30 s at 94°C, 30 s at 60°C and 1 min at 72°C. The PCR products were purified for DNA sequencing using the Wizard PCR Preps DNA Purification System from Promega (Madison, WI). A duplicate PCR from genomic DNA followed by SSCP analysis was always performed to confirm a mutation.
DNA sequencing
DNA was sequenced with the Thermal Sequenase Cycle Sequencing kit from Amersham Life Science (Cleveland, OH) according to the manufacturer's instructions. The sequences of the primers were: 5'-TGT TCA CTT GTG CCC TGA CT-3' and 5'-CAG CCC TGT CGT CTC TCC AG-3' for exon 5, 5'-GCC TCT GAT TCC TCA CTG AT-3' and 5'-TTA ACC CCT CCT CCC AGA GA-3' for exon 6, 5'-AGG CGC ACT GGC CTC ATC TT-3' and 5'-TGT GCA GGG TGG CAA GTG GC-3' for exon 7, 5'-TTC CTT ACT GCC TCT TGC TT-3' and 5'-CGC TTC TTG TCC TGC TTG CT-3' for exon 8. Both strands of DNA and the splicing donor and acceptor sites were sequenced using these primers. The reaction mixture was loaded on a 6% polyacrylamide sequencing gel made from the Sequagel DNA Sequencing Solutions (National Diagnostics, Atlanta, GA). For direct sequencing, the PCR products amplified from genomic DNA were purified and directly used for sequencing.
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Results
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p53 gene mutation in human esophageal SCC
In 43 esophageal SCC, 29 samples had p53 protein accumulation and 25 of these samples had p53 mutations. Fourteen samples had no p53 protein accumulation, five of these had either a deletion, intron mutation or stop codon mutation (Table I
). Taken together, p53 gene mutations were detected in 30 out of 43 (70%) cases of human esophageal SCC specimens. Two of the specimens contained two mutations in each sample and one had three mutations. Among the 34 mutations, there were 11 in exon 5, four in exon 6, seven in exon 7, nine in exon 8, and one each in introns 4, 5 and 6. There were 27 point mutations (14 were G:C to A:T transitions), six deletions and one insertion. Mutations at codon 273 were detected in four of our specimens, and mutations at codons 158, 159, 212, 248 and 282 were each detected in two specimens. Even with the understanding that some gene mutations would not result in p53 synthesis, there was a significant correlation between p53 protein accumulation and p53 gene mutation by Fisher's exact test. Further analysis of the results indicated that all of the 18 samples with `+++' p53 staining (intense staining in almost all the cancer cells) or `++' staining (moderate staining in most of the cancer cells or intense staining in the peripheral regions in 1040% of the cancer nests) contained one or more mutations. On the other hand, carcinomas with `+' staining (weak staining or moderate to intense staining in the peripheral region of <10% of the cancer nests) (10 cases) may not have mutation, or may have different mutations, or no mutation in the nearby BCH or DYS lesions.
Although no mutation was detected in exons 4 and 9 of the p53 gene in esophageal SCC, the frequencies of alleles at the codon 72 polymorphic site in exon 4 were determined to be 11.6, 65.1 and 23.3%, for Arg/Arg, Arg/Pro and Pro/Pro, respectively, in 43 specimens (data not shown). There is no correlation between p53 gene mutation and allelic frequencies at this polymorphic site.
Comparison of p53 mutations at different areas within each primary tumor
p53 gene mutations at different positions from each primary tumor with homogeneous positive p53 immunostaining were compared in five esophageal SCC specimens. Four samples were taken from each pathologically defined tumor at 3, 6, 9 and 12 o'clock positions, and all of them were shown to contain the same mutations (Figure 1
; Table II
).

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Fig. 1. SSCP and DNA sequencing of the p53 gene in human esophageal cancer specimen number 6. Samples were taken at four different positions from a single tumor: (A) 12 o'clock; (B) 3 o'clock; (C) 6 o'clock; and (D) 9 o'clock. P, placental DNA control. Shifted bands in SSCP analysis and a G to A mutation at codon 146 of the p53 gene in DNA sequencing are indicated by arrows.
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Comparison of p53 mutations in areas heterogeneously stained for p53 protein
We analysed three positivenegative pairs of tumor areas, each from the same subject, that were stained heterogeneously for p53 protein (Figure 2A
). One of them had an intron 6 mutation in the area stained negative for p53 protein, whereas the neighboring area stained positive for p53 protein had an additional 3 bp in-frame deletion in exon 7 (Table III
). No p53 mutation was detected in either the p53-positive or -negative areas of the other two pairs of SCC specimens.

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Fig. 2. Immunostaining pattern in SCC. Immunostain-positive and -negative areas are indicated by a closed and an open arrow, respectively. (A) Heterogeneous staining of p53 protein in human esophageal cancer specimen 920937. (B) p53 immunostaining of a well differentiated esophageal cancer nest in specimen 920922. The peripheral area of the cancer nest is stained positive for p53 protein, whereas the center of the cancer nest is stained negative for p53 protein.
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Human esophageal SCC can be classified as well differentiated and poorly differentiated types based on their degree of differentiation. Most poorly differentiated cancers had homogeneous p53 staining. The well differentiated cancers form cancer nests as a result of differentiation and the p53-positive cells were observed mainly in the peripheral area of the cancer nests (Figure 2B
). Three well differentiated cancer nests were selected from sample 920922 which contained a known p53 mutation (G to A mutation at codon 273 in exon 8) in the peripheral p53-positive cells. The centers of these cancer nests which only contained p53-negative cells were microdissected out separately for p53 mutation analysis. No mutation was detected in any of these samples (Table III
).
p53 gene mutation in human esophageal non-cancerous lesions adjacent to SCC
All non-cancerous lesions analysed herein were p53 immunostain-positive lesions. p53 Gene mutations were detected in seven out of 16 (47%) lesions with BCH, eight out of 12 (67%) lesions with DYS and six out of seven (86%) lesions with CIS (Tables I and IV
). The earlier lesions (BCH and DYS) generally had a lower percentage of p53 mutation than the more severe lesions (CIS and SCC). All mutations found in DYS and CIS were the same as those found in the corresponding SCC (Figure 3
; Tables I and IV
). In eight lesions with BCH which contained p53 mutations, three of them (samples 920937, 920972 and 6) were the same as the nearby SCC. In subject 920937, besides the intron mutation found in both BCH and SCC, an additional deletion in exon 7 was also detected in SCC. In subject 920972, in addition to the codon 158 mutation found in both BCH and SCC, a deletion in exon 5 between codon 176 and 182, and a point mutation in exon 8 at codon 273 were also detected in SCC. The other 5 had codon 158 mutations which were different from the nearby SCC (Figures 3 and 4
; Table I
). Four of these five cases had a G to T mutation resulting in the change of Arg to Leu, and the other subject had a G to A mutation resulting in the change of Arg to His.

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Fig. 3. Comparison of SSCP analysis results among lesions with different severities in human esophageal specimens. (A) Exon 5 of the p53 gene. (B) Exon 8 of the p53 gene. P, placental DNA control. Shifted bands are indicated by arrows.
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Fig. 4. DNA sequencing analysis of the p53 gene in BCH and SCC of specimen 920928. The G to A transition at codon 158 in exon 5 was detected in BCH, but not in the nearby SCC (left side). Instead, an 18 bp deletion between codon 176 and 182 was observed in SCC (right side).
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Discussion
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In the present study, p53 gene mutations were detected in 70% SCC specimens from a high-risk population in Henan, China. All mutations were present in exons 58, which encode the DNA binding domain. Based on the updated p53 Gene Mutation Database containing 5961 mutations (18), codons 175, 245, 248, 249, 273 and 282 were identified as mutation hotspots in human cancers. Our results are generally consistent with this pattern as well as the mutation spectrum reported by Lung et al. (19) for esophageal carcinoma samples from Zhengzhou of the Henan Province, China. Codon 273, which was detected in four of our specimens, seems to be a hotspot for human esophageal cancer. Codons 248 and 282 were each detected in two specimens. They all code for the hydrogen-bond-forming amino acid, arginine, which is known to be critical for DNA binding (20). Fourteen out of the 27 point mutations were G:C to A:T transitions, 10 of which were at a CpG site, suggesting that O6-alkyldeoxyguanosine may be a major adduct of DNA alkylation by certain environmental carcinogens. Indeed, a measurable O6-methyldeoxyguanosine level was observed in human esophageal specimens from Linxian (now renamed Linzhou) (21). Alternatively, deamination of 5-methylcytosine at the CpG site of the non-coding strand, for example, by nitric oxide may also result in the observed G:C to A:T mutation. This latter event could occur in esophagitis which occurs frequently in this high risk population (1).
Previous mutation analysis in our laboratory of the esophageal cancer specimens from Linxian (8) showed a 55% (16/29) mutation rate, and that most mutations were detected in exon 5 or 7. The present results suggest that some mutations in exons 6 and 8 may have been missed due to the SSCP conditions used previously (8). In the present study, we used methylmercury hydroxide instead of the previously used formamide to denature DNA in the loading buffer for SSCP analysis. We had better separation of the two DNA strands for exons 6 and 8 of the p53 gene with methylmercury hydroxide. Gels were also run at 8 or 15 W in addition to the 30 W used in the previous study.
In 43 esophageal SCC, 29 samples had p53 protein accumulation, 25 of which had p53 gene mutations. Fourteen samples had no p53 protein accumulation, five of which had either deletion, intron mutation or stop codon mutation. Thus, 35% (5/14) of the specimens which stained negative for p53 protein still had p53 gene mutations and, therefore, >10% (5/43) of the cases containing mutation were missed by p53 immunostaining alone. The other nine samples had no detectable p53 protein accumulation or gene mutation. Taken together, p53 mutation was not detected in 30% of the SCC samples and 21% also lacked p53 protein accumulation. In the cases where p53 protein accumulation is not accompanied by p53 gene mutation, the wild-type (wt) p53 may have been stabilized by p53-binding proteins, such as mdm-2 or human papilloma virus E6. Such binding may result in the loss of p53 tumor suppressor function. In the cases where both p53 protein accumulation and gene mutation were absent, other genetic defects may be responsible for neoplastic transformation.
In our study, most cases of esophageal SCC were shown to have homogeneous p53 immunostaining. Moreover, p53 mutations at different positions of each primary tumor were shown to be the same. This is of great practical importance for our future p53 mutation studies. Only one sample from each tumor needs to be analysed if the tumor is shown to have homogeneous p53 immunostaining. In light of these results and the fact that some lesions with BCH and all of the lesions with DYS or CIS had the same mutations as the nearby SCC, p53 mutation is likely a frequent early event in esophageal carcinogenesis.
The three cases with heterogeneous p53 staining (one of them has an additional p53 mutation in the p53-positive area) may represent a subset of the esophageal SCC in which p53 alteration may be a late event. Alternatively, p53 alteration may still be an early event, but the two tumor areas with heterogeneous immunostaining may be derived from two different clones with different p53 status. However, in a well differentiated cancer nest which is believed to be derived from one clone and formed by differentiation, the failure to detect p53 mutation in the center is surprising. It could have resulted from technical difficulties in analyzing this small area, and this issue remains to be further investigated.
It has been reported that p53 protein accumulation in DYS shares strong similarity with that in cancers based on p53 immunohistochemical analysis (1114,22), and that there is no substantial difference in the cell proliferative activity of DYS and CIS (23). Our findings that the mutations detected in p53 immunostain-positive DYS or CIS were the same as those in the nearby SCC in all cases provide further molecular evidence supporting the notion that DYS is a bona fide precancerous lesion of esophageal SCC. Based on our previous experience, p53 immunostain-positive DYS lesions account for ~80% if not more of the total DYS cases found in the resected tissues.
We found that about half of the p53 immunostain-positive lesions with BCH did not have any p53 gene mutation and, even in samples with mutations, the mutations could be different from those in the nearby SCC. The presence of different p53 mutations in esophageal early lesions and SCC from the same subjects has been reported in a few cases by us and others (5,24). It is likely that in the high-risk population in northern China, the whole esophageal epithelium of an individual is exposed to carcinogens. According to the concept of `field carcerization' lesions may occur independently at different sites, and some of which may eventually progress to cancer. p53 gene mutations occur in esophageal lesions as early as BCH, but only the ones containing mutations with high transforming activities, such as codon 175 and codon 273 mutations, may have the growth advantage to progress eventually to invasive SCC with the acquisition of other genetic changes. Mutations at codon 175 and 273 were shown to have transforming frequencies that were 22- and 8-fold of the basal level with wt p53 protein, respectively (25). The reason why we did not detect any of these mutations in BCH of the resected specimens, but did observe them in biopsy samples (8), is probably due to the possibility that the BCH lesions containing these mutations may have already developed into SCC in the cancerous patients. On the other hand, mutations at other codons such as codon 158 may be less tumorigenic so that the BCH lesions with such mutations are less likely to progress and remain as early lesions. We reason that if these early lesions were to progress to cancer, one would expect to see more of the same mutation in the cancerous lesions. Codon 158 contains a CpG site which is highly sensitive to mutagenesis, but this mutation has been detected in only two esophageal cancer subjects according to the p53 Gene Mutation Database (18). It is possible that this mutation may be a less important event in human esophageal carcinogenesis. In specimens 920925, 920928 and 920972, deletions and hotspot mutations detected in the nearby SCC may have been the driving force of cancer development, despite the presence of codon 158 mutation in BCH. We could not, however, rule out the possibility that these BCH lesions may still be young lesions that could progress to dysplasia or invasive cancer, but this event is apparently much slower than the main carcinogenic events observed in the carcinoma. Further studies are needed to elucidate the relationship between the mutations in BCH and in SCC.
Based on our results the roles of p53 mutation in the development of human esophageal cancer may be conceived as follows: (i) p53 mutation occurs early in esophageal carcinogenesis and is responsible for the loss of cell cycle control. Other genetic changes may accumulate as the lesions progress, and the same p53 mutation is present in the abnormal cells at all stages. It should be noted that only the mutations with strong transforming activity inevitably commit cells to tumorigenesis. (ii) p53 mutations occur late in the carcinogenesis process as a result of selection for additional genetic changes during tumor progression. In this case, the p53 mutations may be different within a primary tumor. This subset of cancers, together with the subset with early p53 mutations, account for ~70% of the total cases analyzed herein. (iii) The other 30% of cases may result from other neoplastic transformation pathways which do not involve p53 mutation. Some of these cases, however, may still have an accumulation of p53 protein (9%) due to stabilization by binding to other proteins.
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Acknowledgments
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Supported by NIH grant CA65871, and facilities from the NIEHS Center grant ES05022 and the Cancer Center Support grant CA72720.
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Notes
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2 To whom correspondence should be addressed Email: csyang{at}rci.rutgers.edu 
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Received August 14, 1998;
revised November 12, 1998;
accepted December 4, 1998.