Download Relationship between Expression of P27, FHIT, PTEN, P73 and

Relationship between Expression of P27, FHIT, PTEN, P73 and
Prognosis in the Esophageal Carcinoma
Y.P. Liu,a
L. Ma,b, *
S.J. Wang,c
B. Liu,a
X. Zhao,a
C.H. Liu,a
M. Han,d
and Y.N. Chena,*
a
Departments of Pathology, bSurgery, and cEndoscopy, Institute of the Forth Hospital of
Hebei Medical University, No.12, Jiankangt Road, Shijiazhuang, 050011, PR China and
d
Department of Biochemistry and Molecular Biology, Institute of Basic Medical University,
No.361, Zhongshan East Road, Shijiazhuang, 050017, PR China
Address correspondence to: YN Chen, MD, The Forth hospital of Hebei Medical Univercity, Jian kang Road,
Shijiazhuang, 050011, PR China. Email: [email protected]
Abstract
AIM: To investigate the differences between the expression of P27, FHIT, PTEN and P73
in esophageal small cell carcinoma (ESCC) and esophageal squamous cell carcinoma
(ESC), and to provide a theoretical basis for clinical treatment and predicting prognosis.
METHODS: Paraffin-embedded tissue blocks of 60 cases of ESCC and 100 cases of ESC
were obtained from the Forth Hospital of Hebei Medical University. The sections were used
for HE and immunhistochemical staining (S-P). The immunological reagents employed
included antibodies against P27, FHIT, PTEN, P73.
RESULTS: The positive rate of P27, FHIT, PTEN had significant differences between
ESCC and ESC (P<0.01). From I~II and III graded ESC to ESCC, the positive expression
of P27 were decreased but the P73 was increased, showing a ladded change (P<0.05). The
expressions of these oncogenes were related to the differentiation, and can be one of the
factors of influencing prognosis.
CONCLUSION: The positive staining rates of tumor tissues for P27, FHIT, PTEN and
p73 were significantly different in ESCC and ESC. The expressions of these oncogenes
were related to the prognostic factor, and thus, it is valuable for clinical treatment and
judging prognosis to detect the expression of P27, FHIT, PTEN and P73 in ESCC.
Key words: esophageal carcinoma, FHIT, PTEN, P27, P73.
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Introduction
The major histological type of the esophageal carcinoma was squamous cell carcinoma
(ESC). Small cell carcinoma of the esophagus (ESCC) was a rare type and the disease
seemed to be more prevalent recently1,2. The two types of carcinoma had little differences
in the location of disease, and clinical symptoms，but only by survival period, there were
significant differences between them3. To further investigate the relationship between the
oncogenes expression of ESCC and ESC and prognosis had the important clinical
significance. Paraffin-embedded tissue blocks of 60 cases of ESCC and 100 cases of ESC
were employed to carry out contrastive experiment. In our work, we not only observed the
detailed histopathology of HE, but also detected the expression of P27, FHIT, PTEN and
P73 immunohistochemically. We obtained the subjective targets (histological differentiation
and type) and objective targets (tumor embolus, lymphatic metastasis, gene expression).
The results showed that the histological differentiation was related to lymphatic metastasis,
tumor embolus, gene expression and survival period, and provided a theoretical basis for
clinical treatment and predicting prognosis.
MATERIALS AND METHODS
Patients Material
During the period of 1986-1996, tumor blocks were obtained from the patients with
primary esophageal carcinoma at the Forth Hospital of Hebei Medical Univercity who had
been treated with curative resectional surgeries. Histopathological classification of tumors
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were done by two experienced pathologists according to World Health Organization
(WHO)4 histological typing of esophageal tumor. Retrospective pathological review of the
tumor revealed 60 cases with ESCC (diagnosed according to histopathology and
neuroendocrinologic staining). None of the patients had received preoperative radiotherapy
and chemotherapy. And survival data of 5 or more years were available on most patients.
The minimum length of follow-up care was 12 months. Follow-up data for 40 cases of
ESCC and 94 cases of ESC could be used for analysis.
Immunohistochemistry and evaluation
Four-μm sections from formalin fixed, paraffin-embeded tissue were moured on
poly-L-lysine-ciated slide. They were air dried and deparaffinized. The sections were
pretreated with citrate buffer (0.01M citric acid, PH6.0) for 20 min at 100°C in a
microwave oven. After blocking non-specific binding sites with normal goat serum in PBS
for 20 min at 37°C, the sections were incubated overnight at 4°C with PTEN, FHIT
(obtained from Beijing zhongshan biotechnology CO., LTD), P27 and P73 (from DAKO).
After rising with PBS, the sections were incubated with biotinylated goat anti-mouse IgG
for 20 min at 37°C followed by incubated with DAB for 10 min at room temperature.
Finally all of these sections were counterstained with hematosylin solution, followed by
cleared and mounted. The primary antibody was omitted and replaced by PBS in the
negative controls.
The sections were microscopically examined by two pathologists without knowing the
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clinical and pathological information. The whole histological sections were scanned at low
magnification to assess for positive areas. Both the intensity (brown color in various
degrees) and the number of positive staining cells were considered for scoring the results.
The degree of immunohistological staining was judged by a semi-quantitative way.
Immunohistochemical staining was classified in the following two groups.
FHIT5，P736
negative, no staining was present or positive staining was detected in <10%
of the cells; and positive, ≥10% of the cells stained positive.
P27
7-9
Staining in <5% of cells were regarded as negative, staining ≥5% of cells were
regarded as positive. At least 20 high-power fields were chosen randomly, and 2000 cells
were counted.
PTEN10 negative, no staining was present or positive staining was detected in <25% of
the cells; and positive, ≥25% of the cells stained positive.
Results
In 60 cases of ESCC , thirty-seven patients were males and 23 females. The mean age was
54.40 years (range 34~75); according to the 1987 UICC standard, 4 tumors (6.7%) were in
the upper esophagus, 36 (60%) were in the middle and 20 (33.33%) were in the lower
esophagus. And the median survival of the patients was 2.6 years. In 100 cases of ESC,
Sixty-four patients were I~II graded and 36 were III graded. Sixty-seven patients were
males and 33 females. The mean age was 52.70 years (range 32~70); 18 tumors (18%) were
in the upper esophagus, 36 (60%) were in the middle and 20 (33.33%) were in the lower
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esophagus. And the median survival of the patients was 4.65 years. There was significant
difference in survival between ESCC and ESC.
The Results of immunhistochemitry
The positive rate of P27, FHIT, PTEN, P73 (Fig1~4) in ESCC and ESC were 5% (3/60),
60% (36/60) 33.33% (20/60), 73.33% (44/60) and 49% (49/100), 84% (84/100), 68%
(68/100) and 51% (51/100), with significant differences between them (P<0.01) (Table1).
In the III-graded ESC and I~II graded ESC, the positive rate expression of FHIT and PTEN
were 80.56% (29/36), 66.67% (25/36) and 85.94% (55/64), 67.19% (42/64), with no
differences between them (P>0.05) (Table2~3). In the III-graded ESC and I~II graded ESC,
the positive rate expression of P27 and P73 were 30.56%, 55.56% and 62.5%, 48.43%.
From I~II and III graded ESC to ESCC, the positive rates of P27 was decreased but the P73
was increased, showing a ladded change (P<0.05). So the expressions of these genes were
related to the differentiation, the degree of the expressions of these genes could suggest the
differentiation of the tumors, and could be one of the factors of influencing prognosis. The
correlation between prognosis and the expression of P27, FHIT, PTEN could be found in
the ESCC, the survival period was shorter with the decreased expressions of these genes.
The survival period of patients with positive expression of these genes was longer than the
ones with negative expressions, and the patients that vessel invasion and lymph node
metastasis were positive had the tendency of low expressions, especially for the expression
of PTEN. The correlation between prognosis and the expression of P73 could be found in
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the ESCC and ESC, the survival period was shorter with the increased expressions of the
gene, and was not related to the sex, age, vessel invasion and lymph node metastasis. The
result suggested that P73 was involved in the development of esophageal carcinoma, and
could be one of the factors predicting the prognosis (Table 2~3).
Discussion
The study of ESCC (derive from mutipotent stem cells11) and ESC further proved that the
vicious degree of ESCC is higher than ESC3. The two type tumor have much differences in
histopathology, differentiation and biological features12, both types may coexist in the same
tumor, the histopathology and differentiation could be different in different areas.
Furthermore, we could not resect all tumor tissue in our work for section, traditional
clinicopathological criteria used to predict clinical outcome are largely inadequate. Thus the
characterization of biological prognostic factors that may help in identifying patients with
different clinical outcome would greatly facilitate management of disease. In our work, we
applied immune staining to detect the expression of P27, FHIT, PTEN and P73 in ESCC
and ESC in order to provide the basis for clinical treatment and predicting prognosis.
Comparison with previous studies theP27 expression and prognosis in cancer
P27 gene is a member of the Cip/Kip family of cycline-dependent kinase inhibitor,
which can associate with cyclinD1-CDK4 and cyclineE-CDK2 complexes and abrogate
their activities. Overexpression of P27 protein in mammalian cells induces a G1 block of
the cell cycle13-15. At present work, the low expression of p27 is correlated with short
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survival period. When the P27 expression is high, the result is contrary16. From I~II, III
graded ESC to ESCC, the positive rate of P27 expression is decreased, with a ladded
change, indicating that the prognosis is poorer with increased grade in esophageal
carcinoma. These results show that, in the development of esophageal carcinoma P27
protein lose their abrogating activities. However, in the cases of decreased expression of
P27 protein, the survival period is shorter, the expression of P27 protein can be a dependent
prognostic of predicting prognostic.
In this study, we present evidence for a role of P27 in patients of esophageal
carcinoma as an independent prognostic predictor of clinical outcome. Patients that show
loss of P27 expreession are at higher risk of poor prognosis and death of disease, suggesting
that P27 to be a prognostic factor correlating with esophageal survival time.
Comparison with previous studies the FHIT expression and prognosis in cancer
Loss of FHIT expression has been reported in a variety of common tumors, including
esophageal carcinoma5, and is associated with exposure to environmental carcinogens. In
our results, the incidence of FHIT protein expression was lower in ESCC than ESC, as is
the case in the lung17. It has been postulated that the FHIT mutation is an early event in
esophageal carcinogenesis, and is related with proliferation and prognosis 18. Loss of FHIT
has been reported to correlate with more aggressive disease in bladder19 or breast20
carcinomas. In our study of esophageal carcinoma, we demonstrated that loss of FHIT
expression was higher in ESCC than in ESC, and was respect to the vessel invasion and
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prognosis.
Comparison with previous studies thePTEN expression and prognosis in cancer
PTEN/MMAC1/TEP1 was recently identified as a tumor suppressor gene located at
10q23.3 and was deleted or mutated in a variety of advanced tumors. PTEN is a
dual-specificity phosphatase with lipid and protein phosphatase activity21, has been shown
to play an important role in the pathogenesis of a variety of human cancers. PTEN gene
product immunoreactivity is consistently absent in primary small cell carcinoma of the
lung22, and this is consistent with our work, suggesting that abnormalities of PTEN gene
may play a role in carcinogenesis. In contrast, 66.7% of ESCC and 32% of ESC were
PTEN-negative. There are significant clinicopathological differences between the cases
with respect to vessel invasion or lymph node metastasis, and that this loss of PTEN is an
independent adverse prognostic factor for disease free survival.
Comparison with previous studies theP73 expression and prognosis in cancer
The structure of P73 protein is highly homogeneous to P5325 and the P73 also shares
some of common functions with P53 protein, such as activating the transcription of other
genes, inhibiting cell growth and inducing apoptosis, indicating that P73 is a P53-like
tumor suppressor23. However, the activation of P73 allele may contribute tumor progression
and the increased levels of P73 mRNA transcription have been found in various tumors
compared to the surrounding normal tissues24. The facts suggest that P73 is rather an
oncogene. In the present study, we found the patients with P73 positive tumors had a short
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survival than those with P73 negative ones in the whole group of patients. Both frequency
and intensity of the P73 expression were markedly increased from normal tissue to primary
tumor and to metastasis, which indicated that P73 might be involved in the development of
esophageal cancer including metastasis. In summary, the overexpression of P73 is a
valuable prognostic marker to predict poor outcome for the patients with esophageal cancer.
Furthermore, the analysis in the combination of multi-genes expressions show that
the patients with positive expression rates of FHIT, PTEN had the longest 5-year survival,
while in ESC, there is not significance of FHIT, PTEN expression between fewer than
5-year survival and more than 5-year survival. The data may suggest that the prognosis of
patients with esophageal carcinoma is not only related to tumor biological behavior and
differentiation, but is related to organism′s gene alteration. Therefore, in the patients with
the tumor of high vicious degree，abnormality of gene structure is the point of biological
pathology.
At present study, the results show that from I-II, III graded ESC to ESCC, the positive
ratios of the lymph node metastasis are increased and the median survival period are
decreased, indicating that the prognosis is poorer with increased grade in esophageal
carcinoma. Thus we can judge the prognosis by the differentiation. In addition, the other
important factor of prognosis is residual end carcinoma, which is affected by the
differentiation of occurent. The differentiation lower, the ratio of residual end carcinoma is
higher. The reason may be that the invasive capacity is increased and genesis of carcinoma
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is multi-point.
Conclusion
At present study, there are significant differences between ESC and ESCC with
biological behavior and gene expression, showing that the high vicious degree of ESC may
contribute to the poor prognosis. But intermediate state may exist between ESC and ESCC,
and determine the differences of gene expression. Abnormalities of one or more gene
expression may represent the biological behavior of ESC worsen. So the detection of
muti-gene expression is significant for predicting the prognosis and directing clinical
treatment.
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Figure and table legends
Fig.1 A, The negative expression of P27 in ESCC (SP×350); B, The positive expression of
P73 in ESCC (SP×350); C,The positive expression of PTEN in ESC, (SP×350); The
negative expression of PTEN in ESCC (SP×350).
Table 1. The gene expression in esophageal carcinoma
Table 2. The relationship between gene expression and biological features in ESC and
ESCC.
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Table 3. The relationship between gene expression and prognosis in ESC and ESCC
Fig. 1
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Table 1
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Table 2
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