834
Mutational Spectra of P53 in Geographically Localized
Esophageal Squamous Cell Carcinoma Groups
in China
Wei Cao, M.D., Ph.D.1,2
Xufeng Chen, Ph.D.3
Huifang Dai, B.S.3,4
Huizhen Wang, R.N.5
Binghui Shen, Ph.D.4
David Chu, M.D.4
Taylor McAfee, Ph.D.2
Zuo-Feng Zhang, M.D.,
Ph.D.
2
1
Department of Pathology, Xinxiang Medical College, Xinxiang, Henan, People’s Republic of China.
2
Department of Epidemiology, University of California–Loc Angeles School of Public Health and
Jonsson Comprehensive Cancer Center, Los Angeles, California.
3
Zhejiang Cancer Research Institute, Hangzhou,
Zhejiang, People’s Republic of China.
4
Department of Radiation Research and Division
of Surgery, City of Hope National Medical Center,
Duarte, California.
5
Department of Medicine, Xinxiang Central Hospital, Xinxiang, Henan, People’s Republic of China.
Supported in part by grant 954020900 from the
Henan Natural Science Foundation and by a SPORE
Lung Cancer Fellowship Award from the National
Institutes of Health (grant P50CA90388).
The authors thank Fubao Chang from the Department of Surgery, Linzhou Esophageal Tumor Hospital and Youhua Jiang from the Division of Surgery, Zhejiang Cancer Hospital for their assistance
in the collection of paired tumor samples and the
follow-up of patients. They also are grateful to Dr.
Shu Zheng and Dr. Jianjian Li for their advice and
support for this study.
Address for reprints: Zuo-Feng Zhang, M.D., Ph.D.,
Professor of Epidemiology, Department of Epidemiology, University of California–Los Angeles
School of Public Health, 71-225 CHS, Box 951772,
Los Angeles, CA 90095-1772; Fax: (310) 2066039; E-mail: [email protected]
Received January 23, 2004; revision received April
21, 2004; accepted May 18, 2004.
BACKGROUND. Esophageal carcinoma is a particularly interesting tumor because of
the dramatic difference in its incidence and geographic distribution among populations of similar ethnic origin. Epidemiologic data have suggested that many
environmental exposures may be associated with an increased risk of its formation.
METHODS. In this study, 92 samples of esophageal squamous cell carcinoma
(ESCC) were collected from patients who resided in 2 geographic areas in China
with different incidences of ESCC: Linxian and Zhejiang. Overexpression and
mutations of the p53 tumor-suppressor gene were examined by using immunohistochemistry, single-strand conformation polymorphism analysis, and direct
sequencing.
RESULTS. The rates of point mutation and overexpression of p53 in the ESCC
specimens studied were 30.4% (29 of 92 specimens) and 51.1% (47 of 92 specimens), respectively. The overexpression of p53 was associated with tumor metastasis and with 5-year case fatality. Significant differences were found in the rates of
overexpression and mutations in patients with clinical T2 tumors between the
specimens from Linxian, which is a high-incidence geographic area, and the
specimens from Zhejiang, which is a low-incidence area. Furthermore, different
mutational spectra were found in the tumor samples from these two geographic
areas: In tumor samples from Linxian, the most common substitution mutation
was a transversion in exon 5, whereas the most common mutations in tumor
samples from Zhejiang were transitions in exon 7.
CONCLUSIONS. The data suggest that the mutation and overexpression of p53 may
play important roles in the development of ESCC. The changes in p53 may reflect
environmental exposure to the different combinations of mutagenic factors and
genetic instability demonstrated by the populations in Linxian and Zhejiang. The
overexpression of p53 protein may have significance as a prognostic factor for
patients with esophageal carcinoma. Cancer 2004;101:834 – 44.
© 2004 American Cancer Society.
KEYWORDS: esophageal squamous cell carcinoma, mutational spectra of P53,
immunohistochemistry, single-strand conformation polymorphism, sequence, p53
protein, high-incidence area, low-incidence area.
E
pidemiologic studies successfully have identified several cancer
risk factors in the human population, including exposure to chemical and physical mutagens (e.g., cigarette smoke, alcohol, heterocyclin amines, asbestos, and ultraviolet radiation), infection by certain
viral and bacterial pathogens, occupation, hormones, and dietary
nongenotoxic constituents (e.g., macronutrients and micronutrients).1– 4 However, the precise molecular mechanisms by which these
influences generate or promote the genetic events required for tu-
© 2004 American Cancer Society
DOI 10.1002/cncr.20437
Published online 23 June 2004 in Wiley InterScience (www.interscience.wiley.com).
P53 in Geographically Localized ESCC in China/Cao et al.
morigenesis have not been delineated. Molecular epidemiology of human malignancy has the challenging
goal of determining the molecular events of interaction between environmental risk factors and genetic
materials that subsequently lead to carcinogenesis.
The focus on risk assessment requires a multidisciplinary strategy to investigate interindividual variation
in cancer risk and gene-environment interactions.
Thus, esophageal carcinoma has become one of the
most interesting tumors among the spectrum of human malignancies in which to pursue molecular studies, because there are dramatic geographic differences
in incidence and distribution among populations of
similar ethnic origin worldwide.5,6 Dietary factors and
cultural habits have been identified in association
with risk differences for this highly fatal disease.7–10
One area of intense interest in recent molecular
epidemiologic studies in esophageal carcinoma has
been the P53 tumor suppressor gene,11,12 which encodes a nuclear phosphoprotein with cancer-inhibiting properties. It is a multifunctional transcription
factor that is involved in the control of cell cycle progression, DNA integrity, and cell survival in cells exposed to DNA-damaging agents.
Loss of p53 activity predisposes cells to the acquisition of oncogenic mutations and may favor genetic
instability. Furthermore, it has been shown that the
P53 gene is one of the genes that is mutated most
frequently in human malignancies, i.e., point mutations in this gene have been found in more tumors
across the spectrum of human malignancies than in
any other gene.13–15 It is noteworthy that approximately 80% of P53 mutations are missense mutations
that lead to amino acid substitutions, subsequently,
that alter the protein conformation and increase the
stability of p53.14 A high frequency of P53 gene mutations has been found in esophageal carcinoma.16 –18
The P53 point mutations in esophageal carcinoma
have been correlated with patients’ endogenous and
exogenous environmental exposures.17,18 However,
further studies of the frequency and timing of onset of
mutations and mutational spectra of P53 should provide insight into the etiology and molecular pathogenesis of esophageal carcinoma and may generate hypotheses for future investigations.19
There are several geographic areas with a high
incidence of esophageal carcinoma, especially in
northern China.20,21 In Linxian, a county in the Henan
province with a population of 800,000, the age-adjusted mortality rates for the incidence of esophageal
carcinoma are up to 169 per 100,000 population.22 The
growing epidemiologic data have implicated several
etiologic possibilities, including nitrosamines, nutritional deficiencies, fermented and moldy foods, and
835
inhalation of polycyclic aromatic hydrocarbons. All of
the above-mentioned factors qualify as environmental
causes of this highly fatal disease in the region.9,20,22,23
In Zhejiang, a province in eastern China, there has
been a low incidence of esophageal carcinoma. According to the malignant tumor mortality survey of
Chinese residents during 1974 –1976, the adjusted
mortality rate for esophageal carcinoma was 10.09 per
100,000 population.24 With regard to etiology, no
study has been conducted on the risk factors for
esophageal carcinoma in this low-incidence area.
However, several studies25–27 have been conducted on
the risk factors for large bowel carcinoma. These risk
factors include low intake of crude fiber, high-lipid
diet, history of intestinal polyps, emotional trauma,
and family history of cancer. These studies suggest
that there are different environmental and genetic
factors at work in Linxian and Zhejiang.
In the current study, the expression of p53 protein
and mutations in the P53 gene in samples of esophageal squamous cell carcinoma (ESCC) from patients
living in Linxian and Zhejiang were studied. Experiments were performed using immunohistochemistry,
single-strand conformation polymorphism (SSCP),
and DNA sequencing. The objectives of this study
were to characterize p53 alterations in the carcinogenesis and development of esophageal carcinoma and to
determine the geographic variations in P53 mutational spectra in this disease.
MATERIALS AND METHODS
Tumor Samples
Overall, 92 patients with ESCC who had undergone
esophagectomy at the Linxian Esophageal Tumor
Hospital (47 patients) between 1979 and 1991 and at
the Zhejiang Cancer Hospital (45 patients) between
1980 and 1990 were included in this study. There were
64 men and 28 women and they ranged in age between 37–71 years (mean age ⫾ standard deviation:
51.3 ⫾ 9.2 years for males and 54.5 ⫾ 7.4 years for
females). The tumors were staged according to the
TNM staging system.28 Data on the survival status
(dead or alive) 5 years after surgery were collected. All
tumor samples were fixed in formalin and embedded
in paraffin. There were 194 tissue blocks, including 92
primary ESCC lesions, 78 matched normal esophageal
mucosa (5–10 cm adjacent to tumor site), and 24
lymph nodes without metastases obtained from the 92
patients. Each block was sectioned serially into 5-␮mthick sections, 1 of which was stained with hematoxylin and eosin for histopathologic analysis. The other
sections were used in the study of p53 protein expression and gene mutation.
836
CANCER August 15, 2004 / Volume 101 / Number 4
Immunohistochemistry
A conventional peroxidase method was used in the
immunohistochemical analysis, as described previously,29 with a modification of microwave oven heating for technical enhancement. The primary antibody
was a 1:200 dilution of a monoclonal mouse antibody,
DO-7, which was raised against an epitope between
amino acids 1 and 45 in the C-terminal domain of
human wild-type and mutant p53 (Pharmingen, San
Diego, CA). In each batch of immunostaining, we used
positive esophageal carcinoma samples as controls.
For negative controls, the primary antibody was replaced with Tris buffered saline. Assessment was carried out according to the intensity of staining in the
nuclei of neoplastic cells, and the results of immunostaining in tissues were scored, with scores of 1 ⫹ indicating weak staining, 2 ⫹ indicating moderate staining, and 3 ⫹ indicating strong or dark-brown staining.
A sample was defined to be positive when ⬎ 10% of
cells demonstrated staining intensity from at least 1
⫹ to 2 ⫹ (weak to moderate).30
DNA Extraction
Paraffin embedded tissue sections were deparaffinized, placed in cell lysis buffer (100 mM Tris-Cl, pH
8.4; 0.5 mM ethylenediamine tetraacetic acid [EDTA];
1% sodium dodecyl sulfate;, and 0.5 mg/mL proteinase K), and incubated at 55 °C overnight. After phenol
and chloroform extractions, DNA was precipitated in
ethanol and resuspended in sterile Tris-EDTA (TE)
buffer (10 mM Tris-Cl, pH 8.0; 1 mM EDTA). Prior to
the polymerase chain reaction (PCR), an aliquot of
DNA solution was examined by agarose gel electrophoresis.
PCR-SSCP
Different regions of exons 5– 8 of the P53 gene were
amplified individually using PCR. PCR amplifications
were performed in 25 ␮L reaction mixture containing
200 –500 ng of genomic DNA and 10 pmol of the following sense and antisense primers, respectively, essentially as described previously31: exon 5, 5⬘-TTC CTC
TTC CTG CAG TAC TCC-3⬘ and 5⬘-GCC CCA GCT GCT
CAC CAT CG-3⬘; exon 6, 5⬘-CAC TGA TTG CTC TTA
GGT CT-3⬘ and 5⬘-AGT TGC AAA CCA GAC CTC AGG3⬘; exon 7, 5⬘-TCT CCT AGG TTG GCT CTG AC-3⬘ and
5⬘-CAA GTG GCT CCT GAG CTG CA-3⬘; and exon 8,
5⬘-CCT ATC CTG AGT AGT GGT AA-3⬘ and 5⬘-GTC
CTG CTT GCT TAC CTC G-3⬘. PCR parameters were as
follows: 1 cycle at 94 °C for 4 minutes followed by 36
cycles at 94 °C for 1 minute, 57 °C for 1 minute, and 72
°C for 1 minute. The reaction was terminated by a
7-minute of extension at 72 °C.
Amplified products were screened for sequence
variations using SSCP analysis, as described previously.32 Briefly, equal volumes of stop solution (96% formamide, 20 mM EDTA, and 0.05% bromophenol blue)
and PCR products (6 ␮L each) were mixed, heat denatured at 98 °C for 5 minutes, and quickly chilled on
ice until loading. Denatured products were electrophoresed on 8% native polyacrylamide gels supplemented with 6% glycerol. Electrophoresis was performed at 50 watts in 0.5 ⫻ Tris-borate-EDTA buffer
for 3 hours at 4 °C. After electrophoresis, gels were
silver stained and photographed.
DNA Sequencing
DNA samples with a mobility shift band present in
SSCP analysis were reamplified using the corresponding primer set and the procedure described above.
Thirty cycles of PCR reactions were employed. PCR
products were purified with QIAquick PCR purification kit (QIAGEN, Valencia, CA), and eluted in 50 ␮L
TE buffer. Sequencing reactions were then performed
in 10 ␮L of the eluted DNA using one of the primers
that was used originally for PCR with a Taq sequence
kit from Perkin Elmer. Automatic sequencing was performed on an ABI 377 Sequencer (Perkin Elmer, Norwalk, NJ).
Statistical Analysis
To evaluate the correlations between p53 overexpression and mutation and clinical variables, we compared p53 overexpression and mutation with age (⬍
60 years vs. ⱖ 60 years), gender (male vs. female),
tumor stage at presentation (T1–T4), lymph node invasion (present vs. absent), and 5-year case fatality
survival (dead vs. alive) using 2-tailed chi-square tests.
Immunohistochemical comparisons of p53 overexpression and mutation with clinical variables between
two geographic areas (Zhejiang and Linxian) and
within each geographic area also were assessed by
chi-square test. The FREQ procedure in SAS software
(SAS Institute Inc., Cary, NC) was used.
RESULTS
Immunohistochemical Analysis
Using the DO-7 monoclonal antibody, nuclear p53
protein was detected in 47 of 92 ESCC samples (51.1%)
(Fig. 1). The correlation of p53 expression with the
clinical data and histopathologic characteristics is
summarized in Table 1. The rate of positive p53 expression was higher (28 of 47 samples; 68.3%) in patients who had lymph node invasion compared with
the rate in patients who did not have lymph node
metastasis (19 of 51 samples; 37.3%; P ⬍ 0.01). A
significant difference in the rate of p53 expression also
P53 in Geographically Localized ESCC in China/Cao et al.
837
were no correlations between the rate of P53 gene
mutation and the variables of age, gender, or tumor
classification. It is worth noting that significantly
higher mutation rates were found in patients who had
lymph node invasion and in patients who died within
5 years after surgery (Table 1).
Geographic Variation of p53 Overexpression and
Mutation
FIGURE 1. Immunohistochemical analysis of p53 expression in esophageal
squamous cell carcinoma cells. Positive immunostaining for p53 protein was
localized in nuclei of tumor cells (arrow). The tumor nuclei were stained,
whereas the adjacent stroma tissue was unstained. The slided, paraffin
embedded tissues were stained with peroxidase-labeled streptavidin-biotin
immunostaining methods using the monoclonal antibody of DO-7 (original
magnification ⫻ 400).
was observed between patients who survived for ⱖ 5
years postoperatively (25 of 60 patients; 41.7%,) and
patients who survived for ⬍ 5 years (20 of 27 patients;
70.1%; P ⬍ 0.01). Conversely, the overexpression of
p53 protein did not differ significantly among patients
with respect to age, gender, or tumor classification.
p53 Mutation in ESCC
Strict criteria were used, and even the slightest shift
from normal mobility of the bands on the SSCP gel
was tentatively identified as a candidate for sequence
variation in the amplified products (Fig. 2). Under
these conditions, band shifts were identified in 34 of
92 tumor samples (37.0%), and 29 mutations were
confirmed in 28 of 92 samples (31.5%) by sequence
analysis (Table 2). Amplified products from three tumor samples that yielded normal mobility patterns
and from three corresponding adjacent normal tissue
samples were sequenced, and no mutations were
found in the sequence of the exon 5– 8 regions.
Twenty-seven mutations were single-base substitutions that resulted in amino acid substitutions or
chain terminations. Among these mutations, 16 were
transversions (59.3%), and 11 were transitions (40.7%).
The other two mutations were single-base insertions
that led to a frameshift in amino acid sequence of the
gene (Table 2). There were a number of mutations
clustered at sequences comprising codons 240 –271,
which lie within 1 of 2 conserved regions required for
binding to simian virus 40 (SV40) T antigen.13 There
The rate of p53 protein overexpression (29 of 47 tumors; 61.7%) was higher in the patients from Linxian
compared with the patients from Zhejiang (18 of 45
tumors; 40.0%; P ⬍ 0.05). Particularly in patients with
T2 tumors, the rate of p53 protein overexpression was
significantly higher in the patients from Linxian (21 of
31 tumors; 67.7%) compared with the patients from
Zhejiang (7 of 21 tumors; 33.3%; P ⬍ 0.05). There was
a correlation between the overexpression of p53 protein and 5-year case fatality among the patients from
Linxian (P ⬍ 0.05). However, no such correlation was
found in the patients from Zhejiang (Table 3). Significant differences also were found in the mutation rates
in patients with T2 tumors between Linxian (15 of 31
tumors; 48.4%) and Zhejiang (3 of 21 tumors; 14.3%; P
⬍ 0.05) (Table 4).
Most of the P53 gene mutations detected in the
tumors from patients residing in Linxian were transversion mutations (12 of 18 mutations; 66.7%). Among
these mutations, 10 of 18 mutations (55.5%) occurred
in exon 5. In contrast, transitions were the major mutation type found in Zhejiang (7 of 11 mutations;
63.6%). The latter mostly occurred in exon 7 of the P53
gene, and 3 were found at codon 260 (Fig. 3). It is
noteworthy that there were four mutations in tumors
from Linxian that occurred at a CpG dimer. No such
mutation was found in tumors from Zhejiang (Table
2). Moreover, P53 mutations in tumors from Linxian
were dominated by GC 3 TA transversions (7 of 18
mutations; 38.9%), whereas the most common P53
mutations in tumors from Zhejiang were AT 3 GC
transitions (4 of 11 mutations; 36.4%) and GC 3 AT
transitions (3 of 11 mutations; 27.3%).
P53 Overexpression and P53 Mutation
There was 69.57% agreement (64 of 92 samples) between p53 overexpression and mutation, with 24 of 92
samples (26.09%) that were positive for both and 40 of
92 samples (43.48%) that were negative for both
among. Statistically, the overexpression of p53 was
associated with mutations in the conserved regions (P
⬍ 0.05); however, no association was found with mutation types or specific locations. Geographically, the
agreement between p53 overexpression and mutation
was 75.5% (34 of 45 samples) and 63.8% (30 of 47
838
CANCER August 15, 2004 / Volume 101 / Number 4
TABLE 1
Correlation of p53 Over Expression/Mutation with Clinical Data and Histopathologic Characteristics
p53 Protein over expression
P53 Mutation (%)
Variable
No.
ⴙ
ⴚ
Positive %
P value
ⴙ
ⴚ
Positive %
P value
Summary
Age
⬍ 60 yrs
⬎ 60 yrs
Gender
Male
Female
Tumor classificationc
T1
T2
T3
T4
Lymph node invasion
Present
Absent
5-yr case fatality
Alive
Dead
92
47
45
51.1
—
29a
64
30.4
—
53
39
31
16
22
23
58.5
41.0
⬎ 0.05
2.74b
15
13
38
26
28.3
33.3
⬎ 0.05
0.267b
64
28
36
11
28
17
56.3
39.3
⬎ 0.05
2.24b
16
12
48
16
25.0
42.9
⬎ 0.05
2.93b
14
52
25
1
6
28
13
1
8
24
12
0
42.9
53.9
52.0
⬎ 0.05
1
18
8
1
13
34
17
0
7.1
34.6
32.0
⬎ 0.05
41
51
28
19
13
32
68.3
37.25
⬍ 0.01d
8.76b
19
9
22
42
46.3
17.6
⬍ 0.01d
8.84b
60
27
25
20
35
7
41.7
70.1
⬍ 0.01d
7.831b
14
13
46
14
23.3
48.1
⬍ 0.05d
5.01b
⫹: Positive: ⫺: negative.
a
Mutations in two codons of P53 were found in one patient.
b
Statistically significant (chi-square test).
c
Classification of primary esophageal carcinoma: T1, invades lamina propria or submucosa; T2, invades muscularis propria; T3, invades adventitia; T4, invades adjacent structures.
d
A significant association was found in the statistical analysis.
samples) in the Zhejiang and Linxian areas, respectively.
DISCUSSION
Tumorigenesis occurs when a cell loses its regulated
growth cycle and clonally expands beyond control.
This requires a series of critical molecular events that
cause the cell to divide and escape from normal proliferative control.33 ESCC may follow this model. This
malignancy is of particular interest, because epidemiologic data suggest that multiple environmental exposures are associated with increased incidence.20 Moreover, its clustered geographic distribution suggests
that specific social, dietary, or heritable characteristics
confined to a particular region are involved in its
development.34,35
Intense research efforts are aimed at identifying
alterations in genes involved in the regulation of cell
growth and differentiation. The product of the P53
gene plays an important role in the negative regulation of cell growth. The wild-type p53 protein binds to
specific DNA sequences as a transcriptional factor that
regulates the expression of particular genes in the cell.
Consequently, it blocks cell progression through the
late G1 phase of the cell cycle.13 Some mutant proteins
fail to block this progression, whereas others can gain
a novel function and actually promote cellular proliferation.36
There are significant geographic differences in the
P53 mutation rates in esophageal carcinoma. Several
investigators have reported P53 mutations in esophageal carcinoma around the world. Audrezet et al. reported P53 mutations in 84% of ESCCs from France.16
Tamura et al. reported P53 mutations in 38% of esophageal carcinomas from Japan.37 Gates et al. and
Gamieldien et al. reported that 67% and 17% of esophageal tumors were associated with P53 mutations in
coastal South Carolina38 and South Africa,39 respectively. Previously, Bennett et al. reported P53 mutations in 50% of tumors from China.29 In the current
study, p53 protein expression and mutations were examined in patients from two separate geographic regions with very different ESCC incidences in China.
Overexpression and mutations of P53 were noted in 47
of 92 tumor samples (51.1%) and 28 of 92 tumor samples (30.4%) from patients with ESCC in Linxian and
Zhejiang, respectively. No obvious differences were
found in the mutation rates between the patients from
these two different geographic areas. This may have
been due in part to the fact that mutation detection
was focused on 1 of 4 conserved regions in the P53
gene: exons 5– 8.36 However, the mutation rate in P53
P53 in Geographically Localized ESCC in China/Cao et al.
839
TABLE 2
P53 Mutations in Patients with Esophageal Squamous Cell
Carcinoma from Different Geographic Areas
FIGURE 2. Results of single-strand conformational polymorphism (SSCP) and
sequencing analysis of P53 mutation in esophageal squamous cell carcinoma.
(A) SSCP analysis of P53 mutation. Amplified DNA fragments from different
exons (5– 8) of the P53 gene from the tumor and matched, normal mucosal
tissues from Patient Z12 were screened for sequence variations using SSCP
analysis. The mobility band shift was present in the polymerase chain reaction
(PCR) product from tumor DNA sample in exon 5 of P53 (arrow), indicating
conformation changes in the DNA fragment (Lanes 1, 3, 5, and 7: matched
normal tissue; Lanes 2, 4, 6, and 8: tumor tissue). (B) Sequence analysis of
paired PCR products from exon 5 of P53 in Patient Z12. The arrow indicates a
nucleotide change in codon 167, which resulted in a change from wild type
(CAG) to mutant (TAG).
was significantly higher in T2 tumors from patients
who resided in Linxian compared with the rate in
tumors from patients who resided in Zhejiang.
In Linxian, which is a high-incidence area of primary ESCC in northern China, 29 of 47 patients
(61.7%) had tumors that were positive for p53 immunohistostaining. This rate was significantly higher
than the rate of 40% (18 of 45 patients) in the patients
from Zhejiang (P ⬍ 0.05), which is a low-incidence
area of China. Overall, p53 overexpression from the
patients from Linxian was higher compared with the
51.4% rate among patients from Guangzhou (a city in
southern China)29,40 and lower than the 87.2% rate
among patients from Linxian in another study.41
These discrepancies in the rate of p53 overexpression
from the same area in different studies may be due to
differences in monoclonal antibody, definition of a
positive sample, and refixation in zinc sulfatemethacarn and microwave-heating methods. Nonetheless, the p53 protein expression rate was highest in
Patienta
Exon
Codon
Mutation
Mutation type
Amino acid change
Z3
Z5
Z11
Zb12
Z16
Z22
Z27
Z29
Z37
Z41
Z42
L2
L3
L6
L8
L13
L14
L25
L27
L30
L32
7
7
5
5
7
8
7
5
7
8
7
8
5
5
7
5
6
5
8
5
5
5
5
8
7
5
7
6
5
260
234
132
167
241
266
260
134
232
275
260
273
164
144
240
145
193
155
270
162
175
179
141
271
244
171
243
196
152
TCC ⬎ TGC
TAC ⬎ TGC
AAG ⬎ AGG
CAG ⬎ TAG
TCC ⬎ TTC
GTA ⬎ GGA
TCC ⬎ TAC
TTT ⬎ TCT
ATC ⬎ GTC
TGT ⬎ TGA
TCC ⬎ TCT
CGT ⬎ TGTb
AAG ⬎ AGAG
CAG ⬎ TAG
AGT ⬎ TGT
CTG ⬎ CAG
CAT ⬎ CTT
ACC ⬎ ACA
TTT ⬎ TGT
ATG ⬎ ATGC
CGC ⬎ CACb
CAT ⬎ CTT
TGC ⬎ TGA
GAG ⬎ GAT
GGC ⬎ TGC
GAG ⬎ TAG
ATG ⬎ ATA
CGA ⬎ AGAb
CCG ⬎ CAGb
Transversion
Transition
Transition
Transition
Transition
Transversion
Transversion
Transition
Transition
Transversion
Transition
Transition
Insertion
Transition
Transversion
Transversion
Transversion
Transversion
Transversion
Insertion
Transition
Transversion
Transversion
Transversion
Transversion
Transversion
Transition
Transversion
Transversion
Ser ⬎ Cys
Tyr ⬎ Cys
Lys ⬎ Arg
Gln ⬎ Stop
Ser ¡ Phe
Val ¡ Gly
Ser ¡ Tyr
Phe ¡ Ser
Ile ¡ Val
Cys ¡ Stop
Silent
Arg ¡ Cys
Frameshift
Gln ¡ Stop
Ser ¡ Cys
Leu ¡ Gln
His ¡ Leu
Silent
Phe ¡ Cys
Frameshift
Arg ¡ His
His ¡ Leu
Sys ¡ Stop
Glu ¡ Asp
Gly ¡ Cys
Glu ¡ Stop
Met ¡ Ile
Silent
Pro ¡ Gln
L33
L39
L41
L42
L45
L46
L47
a
b
Letter prefixes indicate patient origin: Z, Zhejiang; L, Linxian
Mutations occurred at CpG dimer.
Linxian in these studies. These results suggest that the
geographic variation in overexpression of p53 may be
associated with environmental exposure to different
combinations of mutagenic factors and genetic instability.
Spectra of P53 mutations vary geographically in
China. Although it is now clear that changes in specific
DNA sequences lead to cancer, the primary factors
that induce these changes in humans and how they
act remain controversial. Sequence changes in genes
can occur due both to exposure to agents that damage
DNA and to biochemical and enzymatic processes.
Mutations can occur nonrandomly in a given sequence,42 and each mutagen or mutagenic process
produces a characteristic fingerprint of DNA alterations that differ with respect to the nature, location,
and frequency of the alteration in the particular
gene.43,44 Patterns of mutations have been associated
with certain environmental carcinogens.1,45,46 Certain
chemical carcinogens, such as tobacco, are known to
840
CANCER August 15, 2004 / Volume 101 / Number 4
TABLE 3
Geographic Variation in p53 Protein Over Expressiona
Zhejiang
Variable
No.
ⴙ
ⴚ
%
Positive
Summary
Tumor classificationb
T1
T2
T3
T4
Lymph node invasion
Positive
Negative
5-yr case fatality
Alive
Dead
Gender
Male
Female
Age
⬍ 60 yrs
ⱖ 60 yrs
45
18
27
40.0
1
21
22
1
0
7
11
0
1
14
11
1
33.3
50.0
19
26
11
7
8
19
32
8
11
5
34
11
24
21
Linxian
P value
(chi-square)
No.
ⴙ
ⴚ
%
Positive
47
29
18
61.7
⬎ 0.05
13
31
3
6
21
2
7
10
1
46.1
67.7
66.6
57.9
26.9
(4.388)
⬍ 0.05d
22
25
17
12
5
13
21
3
34.4
62.5
(2.109)
⬎ 0.05
28
19
14
15
15
3
19
8
44.1
27.3
(0.983)
⬎ 0.05
30
17
12
6
12
15
50.0
28.6
(2.143)
⬎ 0.05
29
18
Geographic variation
P value
(chi-square)
Chi-square
test
P value
4.333
⬍ 0.05
⬎ 0.05
4.333
⬍ 0.05c
77.3
48.0
(4.24)
⬍ 0.05d
1.768
2.422
⬎ 0.05
⬎ 0.05
14
4
50.0
78.9
(4.01)
⬍ 0.05d
1.500
2.793
⬎ 0.05
⬎ 0.05
20
9
10
8
66.7
52.9
(0.87)
⬎ 0.05
3.27
1.797
⬎ 0.05
⬎ 0.05
19
10
10
8
65.5
55.6
(0.47)
⬍ 0.05d
1.30
2.91
⬎ 0.05
⬎ 0.05
a
Statistical significance was determined by chi-square tests.
Classification of primary esophageal carcinoma: T1, invades lamina propria or submucosa; T2, invades muscularis propria; T3, invades adventitia; T4, invades adjacent structures.
c
A significant difference was found between two group of samples enrolled in the study.
d
A significant association was found in the same group of sample in the statistical analysis.
b
TABLE 4
Geographic Variations of P53 Mutations in Patients with Esophageal Squamous Cell Carcinomaa
Zhejiang
Variable
No.
ⴙ
ⴚ
%
Positive
Summary
Tumor classificationb
T1
T2
T3
T4
Lymph node invasion
Positive
Negative
5-yr case fatality
Alive
Dead
Gender
Male
Female
Age
⬍ 60 yrs
⬎ 60 yrs
45
11
34
24.4
1
21
22
1
0
3
7
1
1
18
15
0
14.3
31.8
19
26
8
3
11
23
32
8
8
3
34
11
24
21
a
Linxian
P value
(chi-square)
No.
ⴙ
ⴚ
%
Positive
47
17
30
(1.80)
⬎ 0.05
13
31
3
1
15
1
42.1
11.5
(5.5)
⬍ 0.001d
22
25
24
5
25.0
37.5
(0.50)
⬎ 0.05
8
3
26
8
23.5
27.3
5
6
19
15
20.8
28.6
Geographic variation
P value
(chi-square)
Chi-square
value
P value
36.1
1.49
⬎ 0.05
12
16
2
7.7
48.4
33.3
6.43
⬍ 0.05c
11
6
11
19
50.0
24.0
(3.42)
⬎ 0.05
0.015
1.36
⬎ 0.05
⬎ 0.05
28
19
11
6
17
13
39.3
31.6
(0.29)
⬎ 0.05
1.198
0.09
⬎ 0.05
⬎ 0.05
(0.02)
⬎ 0.05
30
17
8
9
22
8
26.7
52.9
(3.24)
⬎ 0.05
0.08
1.79
⬎ 0.05
⬎ 0.05
(0.36)
⬎ 0.05
29
18
10
7
19
11
34.5
38.9
(0.09)
⬎ 0.05
1.21
0.46
⬎ 0.05
⬎ 0.05
Statistical significance was determined by chi-square tests.
Classification of primary esophageal carcinoma: T1, invades lamina propria or submucosa; T2, invades muscularis propria; T3, invades adventitia; T4, invades adjacent structures.
c
A significant difference was found between two groups of samples in the study.
d
A significant association was found in the same group of sample in the statistical analysis.
b
P53 in Geographically Localized ESCC in China/Cao et al.
FIGURE 3. Patterns of P53 mutation in esophageal carcinoma samples from
Linxian and from Zhejiang. (A) Mutation locations in the P53 gene. The gray
bars represent mutations that were detected in tumor samples from Linxian,
and the black bars represent mutations that were detected in tumor samples
from Zhejiang. (B) Different types of P53 gene mutations were found in DNA
samples from Linxian and from Zhejiang. Most mutations in the samples from
Linxian (67%; 12 of 18 mutations) were transversions, whereas most mutations
in the samples from Zhejiang (64%; 7 of 11 mutations) were transitions.
generate primarily G-T transversions in esophageal
carcinoma.45 In addition, bronchial explants also can
activate benzo(a)pyrene and N-nitrosodimethylamine
metabolically to form promutagenic DNA adducts.46
The pattern of P53 mutations in our study was notable
in two respects: First, a greater incidence of mutations
was found in T2 tumor samples from the patients who
resided in Linxian compared with the patients who
resided in Zhejiang (Table 4). Second, most of the
mutations detected in the patients from Linxian were
transversions (12 of 18 mutations; 66.7%), and 55.5%
of the mutations were located in exon 5. Among the
patients from Zhejiang, 7 of 11 mutations (63.6%) were
transitions, and most (6 of 11 mutations; 54.5%) were
detected in exon 7 (Fig. 4). Of the 11 mutations found
in the patients from Zhejiang, 3 mutations were located at codon 260, which lies within 1 of 2 conserved
regions that are required for binding to SV40 T antigen.13 There also were four mutations in tumors from
Linxian that occurred at a CpG dimer. This dinucleotide is a frequent site of spontaneous mutations in
which 5-methylcytosine deaminates to thymidine. It is
believed that this event is responsible for many germline mutations that cause human genetic disease.47 In
the current study, the majority of mutations found in
tumors from Linxian were GC 3 TA transversions;
841
whereas, in the tumors from Zhejiang, the most common mutations were GC 3 AT or AT 3 GC transitions,
suggesting that there are different carcinogens in
these two regions in China. Two recent epidemiologic
investigations have shown that nitrosamines, which
are formed in moldy foods, and other environmental
factors were the most likely causes of this disease in
Linxian.20,22 Conversely, in southern China (Zhejiang),
where a high-lipid diet and emotional trauma are possible risk factors for the development of tumors, such
as colorectal carcinoma,25,26 environmental factors
may contribute to P53 mutation, which may lead to
the genesis of esophageal carcinomas.48 Establishing a
specific linkage between the environmental factors
identified in epidemiologic studies and the molecular
alterations identified in the current study will require
further investigation with a larger sample size and
more intensive and complete screening of P53 and
other genes. However, the variations in P53 mutational spectra between these two geographic areas
suggest that different combinations of mutagenic factors or genetic instability in the population in these
two geographic areas contribute to the different incidence of ESCC.
The overexpression of p53 is associated with
5-year case fatality in patients with ESCC. The prognostic significance of p53 aberration in patients with
esophageal carcinoma has been examined recently
with conflicting results. Several groups have proposed
P53 as a significant prognostic indicator in esophageal
carcinoma.41,49 –51 However, Sarbia et al. reported 204
patients in whom p53 accumulation was not associated with shorter survival.52 The current results show
that the overexpression of p53 or mutation in the P53
gene was significantly associated with lymph node
invasion and 5-year case fatality, whereas no associations were found between p53 aberrations and clinicopathologic parameters, including patient age, gender, or tumor classification. Our results imply that P53
mutations may affect the biologic behavior of esophageal carcinoma and that the overexpression of p53
protein may be a useful prognostic factor.
The overexpression of p53 has been associated
with mutation. Recently, Bazan et al.53 reported 57%
agreement between the level of p53 expression and its
genetic mutation status. Our study confirmed that
there was a significant correlation between the expression of p53 and P53 gene mutation in 92 samples of
ESCC, with an agreement of 69.57% (64 of 92 samples).
Specifically, statistical analysis of our data also revealed that there was a significant association between
overexpression and mutation of P53 in these 2 geographic areas (P ⬍ 0.01 in Zhejiang; P ⬍ 0.05 in Linxian). These results are consistent with previous re-
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CANCER August 15, 2004 / Volume 101 / Number 4
FIGURE 4. Differences in P53 mutation spectra between samples of esophageal carcinoma from Linxian and from Zhejiang. (A) Mutation spectrum analysis.
Numbers on the left (3, 0, and ⫺ 3) indicate the number of mutations detected in the study. The number at right on the bar (393) is the codon number. (B) Schematic
representation of the functional motifs of p53 protein. The region encoded by exons 5– 8 is located in a central region of P53, which contains 4 of 5 conserved
regions and the DNA binding motif of p53 protein. This central region also functions as a protein-binding domain interacting with simian virus 40 (SV 40) T antigen
and the cellular proteins 53BP1 and 53BP2 (see May and May13). Values in parentheses are P53 codon numbers.
ports.54,55 Moreover, the agreement was slightly higher
in Zhejiang (75.5%) than in Linxian (63.8%), which
may have been due to fewer silent and nonsense mutations in Zhejiang (1 silent and 2 stop codons) compared with Linxian (2 silent, 2 frameshift, and 2 stop
codons). Nine samples with nonsense or frameshift
mutations displayed P53 gene mutation by PCR-SSCP
without p53 protein overexpression. These mutations
may lead either to the production of a truncated protein or to no protein expression.53 In the current study,
most of the mutations occurred in exon 5 in Linxian.
One study reported56 that mutations in exon 5 resulted in low-intensity immunostaining, which may
be related to a p53 protein with a shorter half-life or an
altered conformation. Therefore, there was a slightly
higher concordance in Zhejiang. The discrepancies
between P53 gene mutation and p53 protein overexpression may indicate other potential etiologic factors,
such as human papillomavirus (HPV) infection.
HPV-16 E6 protein has a much higher affinity for p53
and inhibits p53 DNA binding.57 The complex with E6
and p53 may inactivate functions of P53 gene, affecting overexpression of p53. We did not analyze the
mutations outside the exon 5– 8 area; therefore, some
of the samples with P53 gene mutations and null
reactivity for p53 immunostaining may be explained
by mutations occurring in these sites. The samples
with overexpression of p53 protein and no mutation
may be due to the stabilization of wild-type p53.
Cripps et al.58 found several tumors with strong p53
accumulation but without mutations in the gene-coding region, and their results supported the aforementioned view.
The current results must be interpreted in the
context of certain limitations. The human P53 gene is
comprised of 11 exons.13 Thus, because this study only
examined P53 mutations in exons 5– 8, we may have
underestimated the P53 mutation rates, because exons 1– 4 and 9 –11 were not included. Some studies59
have suggested that most P53 mutations (90%) occur
in exons 5– 8 and are mainly missense mutations that
result in increased protein stability. Greenblatt et al.60
reported that only 2% of P53 mutations were in regions outside of the conserved regions of exons 5– 8 in
patients with head and neck carcinoma; moreover,
those mutations frequently were of the nonsense or
frameshift types, which are unlikely to play a major
role in oral carcinogenesis.61,62 In ESCC, mutations
were found rarely in the regions flanking exons 5– 8,63
whereas mutations in the regions outside exons 5– 8
appear to be uncommon.64 Given this background, we
examined P53 mutations only in exons 5– 8.
Another concern was the sample size of the study.
In the current study, we were not interested in comparing tumor classification in the 4 groups; therefore,
we stratified on tumor classification and evaluated the
mutation rates of p53 protein in the different tumor
classifications from the 2 geographic areas. Table 4
shows that, in Zhejiang, 1 patient had a T1 tumor and
22 patients had T3 tumors; and, in Linxian, 13 patients
had T1 tumors and 3 patients had T3 tumors. Such
small sample sizes prevented us from comparing p53
changes in patients with T1 and T3 tumors from the 2
geographic areas. Although this study had the limitation of a small sample size, we did find an association
in patients with T2 tumors. Obviously, this limitation
should be considered in interpreting our findings.
In conclusion, the data presented here imply that
P53 mutations occur commonly in Chinese patients
with esophageal carcinoma and are associated with
P53 in Geographically Localized ESCC in China/Cao et al.
the biologic behavior of esophageal carcinoma. Epidemiologic studies in these two regions in China also
suggest that different combinations of mutagenic factors may contribute to the different incidence and
mutation pattern of P53 in ESCC. It is possible that the
overexpression of p53 protein may be used as a prognostic factor for patients with esophageal carcinoma.
22.
REFERENCES
23.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Minamoto T, Mai M, Ronai Z. Environmental factors as
regulators and effectors of multi-step carcinogenesis. Carcinogenesis. 1999;20:519 –527.
Coleman MP, Esteve J, Damiecki P, Arslan A, Renard H.
Trends in cancer incidence and mortality. Lyon: IARC Scientific Publications, 1993.
Henderson BE, Feigelson HS. Hormonal carcinogenesis.
Carcinogenesis. 2000;21:427– 433.
Hill MJ. Diet, physical activity and cancer risk. Public Health
Nutr. 1999;2:397– 401.
National Institutes of Health. SEER cancer statistics review,
1973–1991 (NIH publication no. 94-2789). Bethesda: National Cancer Institute, 1994.
World Health Organization. Cancer: causes, occurrence and
control. IARC scientific publication 100. Lyon: International
Agency for Research on Cancer, 1990.
Yu MC, Garabrant DH, Peters JM, Mack TM. Tobacco, alcohol, diet, occupation, and carcinoma of the esophagus. Cancer Res. 1988;48:3843–3848.
Sons HU. Etiologic and epidemiologic factors of carcinoma
of the esophagus. Surg Gynecol Obstet. 1987;165:183–190.
Cheng KK, Day NE. Nutrition and esophageal cancer. Cancer
Causes Control. 1996;7:33– 40.
Ghadirian P, Vobecky J, Vobecky JS. Factors associated with
cancer of the esophagus: an overview. Cancer Detect Prev.
1988;11:225–234.
Caron DF, Soussi T. TP53 tumor suppressor gene: a model
for investigating human mutagenesis. Genes Chromosomes
Cancer. 1992;4:1–15.
Hollstein MC, Peri L, Mandard AM, et al. Genetic analysis of
human esophageal tumors from two high incidence geographic areas: frequent p53 base substitutions and absence
of Ras mutations. Cancer Res. 1991;51:4102– 4106.
May P, May E. Twenty years of p53 research: structural and
functional aspects of the p53 protein. Oncogene. 1999;18:
7621–7636.
Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers. Science. 1991;253:49 –53.
Harris CC. 1995 Deichmann Lecture—p53 tumor suppressor
gene: at the crossroads of molecular carcinogenesis, molecular epidemiology and cancer risk assessment. Toxicol Lett.
1995;82/83:1–7.
Audrezet MP, Robaszkiewicz M, Mercier B, et al. TP53 gene
mutation profile in esophageal squamous cell carcinomas.
Cancer Res. 1993;53:5745–5749.
Montesano R, Hollstein M, Hainaut P. Genetic alterations in
esophageal cancer and their relevance to etiology and
pathogenesis: a review. Int J Cancer. 1996;69:225–235.
Puisieux A, Lim S, Groopman J, Ozturk M. Selective targeting
of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res. 1991;51:6185–
6189.
Bennett WP, Hussain SP, Vahakangas KH, Khan MA, Shields
PG, Harris CC. Molecular epidemiology of human cancer
20.
21.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
843
risk: gene-environment interactions and p53 mutation spectrum in human lung cancer. J Pathol. 1999;187:8 –18.
Yang CS. Research on esophageal cancer in China: a review.
Cancer Res. 1980;40:2633–2644.
Li L, Lu F, Zhang S. Analysis of cancer modality and distribution in China from year 1990 through 1992—an epidemiologic study. Zhonghua Zhong Liu Za Zhi. 1996;18:403– 407.
Li JY. Epidemiology of esophageal cancer in China. Natl
Cancer Inst Monogr. 1982;62:113–120.
Zhen YZ, Xu YM, Liu GT, et al. Mutagenicity of Alternaria
alternata and Penicillium cyclopium isolated from grains in
an area of high incidence of esophageal cancer—Linxian,
China. In: O’Neill IK, Chen J, Bartsch H, editors. Relevance
to human cancer of N-nitroso compounds, tobacco and
mycotoxins. (IARC Sci Pub 105). Lyon: IARC Scientific Publications, 1991:253–257.
The Office of Cancer Prevention and Treatment Research in
Chinese Department of Public Health. Chinese survey for
malignant tumor mortality. Beijing: The Publisher of Public
Health, 1980.
Chen K, Cai J, Liu XY, Ma XY, Yao KY, Zheng S. Nested
case-control study on the risk factors of colorectal cancer.
World J Gastroenterol. 2003;9:99 –103.
Chen K, Qiu JL, Zhang Y, Zhao YW. Meta analysis of risk
factors for colorectal cancer. World J Gastroenterol. 2003;9:
1598 –1600.
Wang X, Lei T, Ma X. Colon cancer risk factors in Jiashan
County, Zhejiang province, the highest incidence area in
China. Zhonghua Zhong Liu Za Zhi. 2001;23:480 – 482.
Wood DE. Esophageal carcinoma. Manual of clinical oncology, 7th ed. New York: John Wiley & Sons, Inc., 1999.
Bennett WP, Hollstein MC, He A, et al. Archival analysis of
p53 genetic and protein alterations in Chinese esophageal
cancer. Oncogene. 1991;6:1779 –1784.
Nezhat F, Cohen C, Rahaman J, Gretz H, Cole P, Kalir T.
Comparative immunohistochemical studies of bcl-2 and
p53 proteins in benign and malignant ovarian endometriotic cysts. Cancer. 2002;94:2935–2940.
Hollstein MC, Metcalf RA, Welsh JA, Montesano R, Harris
CC. Frequent mutation of the p53 gene in human esophageal cancer. Proc Natl Acad Sci USA. 1990;87:9958 –9961.
Spinardi L, Mazars R, Theillet C. Protocols for an improved
detection of point mutations by SSCP. Nucleic Acids Res.
1991;19:4009.
Fearon ER, Vogelstein B. A genetic model for colorectal
tumorigenesis. Cell. 1990;61:759 –767.
Day NE. Cancer epidemiology and prevention, 1st ed. Philadelphia: WB Saunders, 1982.
Parkin DM, Pisani P, Ferlay J. Estimates of the worldwide
incidence of eighteen major cancers in 1985. Int J Cancer.
1993;54:594 – 606.
Chen PL, Chen YM, Bookstein R, Lee WH. Genetic mechanisms of tumor suppression by the human p53 gene. Science. 1990;250:1576 –1580.
Tamura G, Maesawa C, Suzuki Y, Satodate R, Ishida K, Saito
K. p53 Gene mutations in esophageal cancer detected by
polymerase chain reaction single-strand conformation polymorphism analysis. Jpn J Cancer Res. 1992;83:559 –562.
Gates CE, Reed CE, Bromberg JS, Everett ET, Baron PL.
Prevalence of p53 mutations in patients with squamous cell
carcinoma of the esophagus. J Thorac Cardiovasc Surg. 1994;
108:148 –152.
844
CANCER August 15, 2004 / Volume 101 / Number 4
39. Gamieldien W, Victor TC, Mugwanya D, et al. P53 and p16/
CDKN2 gene mutations in esophageal tumors from a highincidence area in South Africa. Int J Cancer. 1998;78:544 –
549.
40. Bennett WP, Hollstein MC, Metcalf RA, et al. P53 mutation
and protein accumulation during multistage human esophageal carcinogenesis. Cancer Res. 1992;52:6092– 6097.
41. Wang DY, Xiang YY, Tanaka M, et al. High prevalence of p53
protein overexpression in patients with esophageal cancer
in Linxian, China and its relationship to progression and
prognosis. Cancer. 1994;74:3089 –3096.
42. Thilly WG. Mutational spectrometry in animal toxicity testing. Annu Rev Pharmacol Toxicol. 1990;30:369 –385.
43. Horsfall MJ, Gordon AJ, Burns PA, Zielenska M, Van der Vliet
GM, Glickman BW. Mutational specificity of alkylating
agents and the influence of DNA repair. Environ Mol Mutagen. 1990;15:107–122.
44. Denissenko MF, Pao A, Tang M, Pfeifer GP. Preferential
formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in P53. Science. 1996;274:430 – 432.
45. Brash DE. Sunlight and the onset of skin cancer. Trends
Genet. 1997;13:410 – 414.
46. Coulondre C, Miller JH, Farabaugh PJ, Gilbert W. Molecular
basis of base substitution hotspots in Escherichia coli. Nature. 1978;274:775–780.
47. Cooper DN, Krawczak M. The mutational spectrum of single
base-pair substitutions causing human genetic disease: patterns and predictions. Hum Genet. 1990;85:55–74.
48. Lung ML, Chan WC, Zong YS, et al. P53 mutational spectrum
of esophageal carcinomas from five different geographical
locales in China. Cancer Epidemiol Biomarkers Prevent.
1996;5:277–284.
49. Shimaya K, Shiozaki H, Inoue M, et al. Significance of p53
expression as a prognostic factor in esophageal squamous
cell carcinoma. Virchows Arch A Pathol Anat Histopathol.
1993;422:271–276.
50. Furihata M, Ohtsuki Y, Ogoshi S, Takahashi A, Tamiya T,
Ogata T. Prognostic significance of human papillomavirus
genomes (type-16, -18) and aberrant expression of p53 protein in human esophageal cancer. Int J Cancer. 1993;54:226 –
230.
51. Coggi G, Bosari S, Roncalli M, et al. P53 protein accumulation and p53 gene mutation in esophageal carcinoma. A
molecular and immunohistochemical study with clinicopathologic correlations. Cancer. 1997;79:425– 432.
52. Sarbia M, Porschen R, Borchard F, Horstmann O, Willers R,
Gabbert HE. P53 protein expression and prognosis in squa-
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
mous cell carcinoma of the esophagus. Cancer. 1994;74:
2218 –2223.
Bazan V, Migliavacca M, Tubiolo C, et al. Have p53 gene
mutations and protein expression a different biological significance in colorectal cancer? J Cell Physiol. 2002;191:237–
246.
Bernardini S, Adessi GL, Billerey C, et al. Immunohistochemical detection of p53 protein overexpression versus
gene sequencing in urinary bladder carcinomas. J Urol.
1999;162:1496 –1501.
Veloso M, Wrba F, Kaserer K, et al. p53 gene status and
expression of p53, mdm2, and p21Waf1/Cip1 proteins in
colorectal cancer. Virchows Arch. 2000;437:241–247.
Esrig D, Spruck CH III, Nichols PW, et al. P53 nuclear protein accumulation correlates with mutations in the p53
gene, tumor grade, and stage in bladder cancer. Am J Pathol.
1993;143:1389 –1397.
Lechner MS, Laimins LA. Inhibition of p53 DNA binding by
human papillomavirus E6 proteins. J Virol. 1994;68:4262–
4273.
Cripps KJ, Purdie CA, Carder PJ, et al. A study of stabilization
of p53 protein versus point mutation in colorectal carcinoma. Oncogene. 1994;9:2739 –2743.
Levine AJ, Momand J, Finlay CA. The p53 tumor suppressor
gene. Nature. 1991;351:453– 456.
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– 4878.
Kannan K, Munirajan AK, Krishnamurthy J, et al. Low incidence of p53 mutation in betel quid and tobacco chewingassociated oral squamous carcinoma from India. Int J Oncol.
1999;15:1133–1136.
Hsieh LL, Wang PF, Chen IH, et al. Characteristics of mutation in the p53 gene in oral squamous cell carcinoma associated with betel quid chewing and cigarette smoking in
Taiwanese. Carcinogenesis. 2001;22:1497–1503.
Vos M, Adms CH, Victor TC, Van Helden PU. Polymorphisms and mutation found in the regions flanking exon 5 to
8 of the TP53 gene in a population at high risk for esophageal cancer in South Africa. Cancer Genet Cytogenet. 2003;
140:23–30.
Wagata T, Shibagaki I, Imamura M, 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 – 850.