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

Relationship between expression of P27, FHIT, PTEN, P73 and
prognosis in esophageal squamous cell carcinoma
Running title: P27, FHIT, PTEN and P73 expression in esophageal squamous cell
carcinoma
Key words: esophageal squamous carcinoma, FHIT, PTEN, P27, P73.
Abstract
To investigate the significance between the expression of P27, FHIT, PTEN and P73 in
esophageal squamous cell carcinoma (ESCC), paraffin-embedded tissue blocks of 200
cases were obtained from the Fourth 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 and P73. From I~II and III graded
ESCC, the positive expression of P27 were decreased but the P73 was increased, showing a
YANNING CHEN, XIAOLING WANG, LI MA, LINGLING ZHANG and YUEPING
LIU
Departments of Patholog, Institute of the Fourth Hospital of Hebei Medical University,
No.12, Jiankangt Road, Shijiazhuang, 050011, PR China
Correspondence to: Dr Yueping Liu, Departments of Patholog, Institute of the Fourth
Hospital of Hebei Medical University, No.12, Jiankangt Road, Shijiazhuang, 050011, PR
China. Email: [email protected]
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. 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.
Introduction
Esophageal carcinoma is a highly aggressive carcinoma with a poor long-term outcome,
and surgery is considered the best option for cure. However, even after R0 resection,
long-term survival is only about 25% in most Western series1. Based on reports from China,
the 5-year survival is reported to be around 35%2. The major histological type of the
esophageal carcinoma was squamous cell carcinoma (ESCC). To further investigate the
relationship between the oncogenes expression of ESCC and prognosis had the important
clinical significance ， paraffin-embedded tissue blocks of 200 cases of ESCC 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
factors
(histological
differentiation and type) and objective factors (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.
2
MATERIALS AND METHODS
Patients Material
During the period of 2002-2007, tumor blocks were obtained from the patients with
primary esophageal carcinoma at the Fourth Hospital of Hebei Medical Univercity who had
been treated with curative resectional surgeries. Histopathological classification of tumor
was done by two experienced pathologists according to World Health Organization (WHO).
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 188 cases of ESCC 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
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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
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.
FHIT3，P734
negative, no staining was present or positive staining was detected in <10%
of the cells; and positive, ≥10% of the cells stained positive.
P275-7 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.
PTEN8 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 200 cases of ESCC, 128 patients were I~II graded and 72 were III graded. 134 patients
were males and 66 females. The mean age was 52.70 years (range 32~70); 36 tumors (18%)
were in the upper esophagus, 120 (60%) were in the middle and 44 (22%) were in the lower
esophagus. And the median survival of the patients was 4.65 years. There was significant
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difference in survival between ESCC and ESC.
The Results of immunhistochemitry
The positive rate of P27, FHIT, PTEN, P73 (Fig1) in ESCC were 49% (98/200), 84%
(168/200), 68% (136/200) and 51% (102/200), with significant differences between them
(P<0.01) (Table1). In the III-graded ESCC and I~II graded ESCC, the positive rate
expression of FHIT and PTEN were 80.56% (58/72), 66.67% (50/72) and 85.94%
(110/128), 67.19% (84/128), with no differences between them (P>0.05) (Table2~3). In the
III-graded ESCC and I~II graded ESCC, the positive rate expression of P27 and P73 were
30.56%, 55.56% and 62.5%, 48.43%. From I~II and III graded 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 the ESCC and ESC, the survival period was
shorter with the increased expressions of the gene, and was not related to the sex, age,
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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 1~3).
Table 1. The gene expression in esophageal carcinoma
Gene expression
n
ESC prognosis (year)
T
P
FHIT
-
30
4.26±1.85
+
158
5.76±2.06
-
62
5.32±2.27
+
126
4.33±2.01
-
94
4.08±2.05
+
94
5.23±2.98
-
92
4.85±2.01
+
96
4.48±2.06
0.87
>0.05
1.54
>0.05
1.90
>0.05
0.56
<0.05
PTEN
P27
P73
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Table 2.
The relationship between gene expression and biological features in ESCC.
Biological
feature
FHIT
P
-
+
24
16
PTEN
-
+
34
6
P
P27
-
P
+
P73
-
+
2
38
30
50
24
30
8
58
P
Vessel
invasion
+
<0.01
24
56
+
30
44
-
18
-
38
<0.01
46
34
58
16
2
>0.05
>0.05
76
4
72
2
Lymph node
Metastasis
>0.05
28
22
24
>0.05
>0.05
<0.01
42
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Table 3. The relationship between gene expression and prognosis in ESCC
gene
ESCC(Ⅰ-Ⅱ)
ESCC(Ⅲ)
P
P27
-
52
50
+
76
22
-
66
32
+
62
40
<0.01
P73
PTEN
7
<0.05
-
44
22
+
84
50
-
18
14
+
110
58
<0.01
FHIT
<0.01
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 ESCC, (SP×350); The
negative expression of PTEN in ESCC (SP×350).
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Discussion
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
cycle9-11. At present work, the low expression of p27 is correlated with short survival period.
When the P27 expression is high, the result is contrary12. From I-II, III graded 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.
Loss of FHIT expression has been reported in a variety of common tumors, including
esophageal carcinoma3, and is associated with exposure to environmental carcinogens. It
has been postulated that the FHIT mutation is an early event in esophageal carcinogenesis,
and is related with proliferation and prognosis13. Loss of FHIT has been reported to
correlate with more aggressive disease in bladder14 or breast15 carcinomas. In our study of
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esophageal carcinoma, we demonstrated that loss of FHIT expression was respect to the
vessel invasion and prognosis.
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 activity16, 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
lung17, and this is consistent with our work, suggesting that abnormalities of PTEN gene
may play a role in carcinogenesis. 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.
The structure of P73 protein is highly homogeneous to P5318 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
suppressor19. 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
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
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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,
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-IIgraded to III graded 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
is multi-point.
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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 ESCC, (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 ESCC.
Table 3. The relationship between gene expression and prognosis in ESCC
14