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Prognostic Value of Caludin-4, nm23-H1 and MIB-1 in
Undifferentiated Nasopharyngeal Carcinoma
Nashwa M. Emara*, Ahlam A. Abd El-Maksoud*, Enas Ibrahim*, Ahmed M.
Zeidan**, and Akram M. Nouh***
Pathology *& General Surgery** Departments; Faculty of Medicine, Benha
University & National Cancer Institute; Cairo University***
Abstract: Nasopharyngeal cancer (NPC) is a tumor of epithelial origin
with complex etiology, and it is one of the most confusing, commonly
misdiagnosed and poorly understood diseases. Currently the standard
treatment of NPC is radiotherapy, but therapy failure is quite common,
making radioresistance an important issue. Claudin-4 is a major tight
junction protein; that regulate the integrity and function of tight junctions.
Aberrant expression of claudin-4 has been shown in various carcinomas
with diverse prognostic implications. Ki67 as a maker of cell proliferation
and nm23-H1as a metastasis marker have been widely used by many
studies to indicate their close relationship with the progression and
prognosis in many tumors. However, little is known about their
prognostic values in undifferentiated NPC.
Aim: The aim of this study is to evaluate the immunohistochemical
staining of claudin-4, nm23-H1 protein and Ki-67 in human
undifferentiated NPC, and the relationship between claudin-4, nm23-H1
and ki-67 expressions and both metastasis and prognosis of patients of
undifferentiated NPC.
Results: Results were correlated with sex, age, extent of tumor, lymph
node status, the presence or absence of distant metastasis and patient
survival. Low claudin-4 expression, low nm23-H1 and high ki-67
expression were associated with distant metastasis and poor survival.
Conclusion: Claudin-4 may be a novel biomarker for prediction of
distant metastasis and unfavorable prognosis in NPC, especially when
combined with nm23-H1 and ki-67.
Key Words: Ki67, nm23-H1, claudin-4 expression, NPC, prognosis.
Introduction:
NPC is an Epstein-Barr virus-related cancer with a high prevalence
in Southeast Asia, Southern China, and North Africa. It has a high
metastatic potential compares to other head and neck cancers. More than
half of the patients present with detectable metastases in the regional
lymph nodes or distant organs at diagnosis (Tsang et al; 2003, Jemal et
al; 2011, Xuebing et al; 2012). NPC is notorious for its potential to
metastasize at the early stages of the disease. For the NPC patients who
have a distant metastasis, the outcome was generally ominous (Xuebing
et al; 2012, Xu et al; 2015, Wang et al; 2015).
Nasopharyngeal carcinoma (NPC) is a squamous cell carcinoma
(Chan et al; 2005) and is subdivided into non-keratinizing and
keratinizing squamous cell carcinomas. NPC accounts for 3.7% of upper
aerodigestive tract carcinomas (Parkin et al; 2001). According to the
tumor-node-metastasis staging system setted by the American Joint
Committee on Cancer, patients are designated into stages 0, I, IIA, IIB,
III, IVA, IVB and IVC (Cho; 2007).
The etiology of NPC includes viral, genetic, and environmental
factors. But the molecular mechanism of the development and
progression of NPC remains poorly understood (Hsueh et al 2010).
Identification of NPC metastasis-associated factors is helpful not only for
understanding the mechanisms involved in NPC metastasis, but also for
finding biomarkers for prognosis and potential therapeutic targets of NPC
and hence may lead to more effective treatment of NPC patients (Liu et
al; 2008 , Xuebing et al; 2012).
Tight junctions (TJs) are specialized regions of cell-cell contact
and have crucial roles in maintenance of cell polarity, adhesion, cellular
arrangement, and paracellular permeability. Claudins have been recently
identified as structural and functional components of the TJs in epithelial
and endothelial cells and shown to play an important role in TJ function.
They are a family of 24 proteins, and various claudins are expressed in
different epithelial cells. Most tissues express multiple claudin proteins,
which is thought to account for the selective variability of different tissue
functions (Van Itallie & Anderson; 2006, Hewitt et al; 2006, Krause et
al; 2007).
Claudins have been of interest in cancer research. It has been
hypothesized that changes and/or loss of claudin expression may play an
important role in tumorigenesis and tumor progression, and altered
expression of claudins has been reported in a variety of human
neoplasms, including colorectal, breast, ovarian, pancreatic, prostate,
tongue and esophageal carcinomas and in rectal well-differentiated
endocrine neoplasms (Kojima et al; 2010). Although the expression
pattern and prognostic value of claudins have been studied in many
carcinomas, to the best of our knowledge, they have not been extensively
investigated in NPC especially undifferentiated phenotype.
Nucleoside diphosphate kinases, a highly conserved family in
eukaryotes, are encoded by NM23 genes. NM23-H1 was identified by
Steeg et al (1988a, b), based on its low expression in metastatic cells.
Low expression or mutation of NM23-H1 has been implicated in cancer
prognosis or metastasis in a variety of tumours and malignant
transformation (Leone et al, 1991; Yih et al, 2002; Fan et al, 2003; Zhao
and Li, 2004; Ma et al, 2005). Exogenous overexpression of the
metastasis suppressor gene Nm23-H1 reduces the metastatic potential of
multiple types of cancer cells and suppresses in vitro tumor cell motility
and invasion (Horak et al; 2007). However, no definite data regarding
NM23-H1 expression in the aggressive undifferentiated NPC phenotype.
Ki-67 is a nuclear proliferation antigen that is expressed in
proliferating cells during the G1, S, G2 and M phases of the cell cycle. Its
expression is used as a marker of cell proliferation. Many studies have
confirmed in a variety of human malignant tumors the relation between a
high Ki-67 LI and poor prognosis. However, other studies, in prostate
cancer and carcinomas of head and neck have shown no correlation
between Ki-67 index and prognosis (őzel et al; 2004). Also, their results
in nasopharyngeal carcinoma were debatable.
The aim of this study is to evaluate the expression of claudin-4
protein in undifferentiated NPC patients and to correlate the results with
clinicopathologic variables, other suggested markers; as nm23-H1 and
Ki67 and disease outcome.
 Material and Methods:
 Human Tissue sampling and immunohistochemical analysis
This retrospective study was carried upon 50 cases diagnosed as
undifferentiated nasopharyngeal carcinoma during the years (2000-2010).
Archival formalin-fixed, paraffin embedded blocks of tumor specimens,
were collected from Pathology Department, Faculty of Medicine, Benha
University and National Cancer Institute, Faculty of Medicine, Cairo
University.
The corresponding H&E slides and the clinicopathological data were
retrieved from the patient archives and reviewed for confirmation of
diagnosis and adequacy of materials.
Follow up of only 22 patients was obtained for 5 years or until the
time of death.
From each block, 4 sections of 4 µm thickness were cut. One section
was used for histopathological study by conventional H&E stain and the
other 3 sections were cut on positively charged slides for immunehisochemical study.
The histopathological classification of the tumor samples was
based on Pathology and Genetics of Head and Neck Tumors" by World
Health Organization in 2005 (Chan et al; 2005). Stage was defined
according to American Joint Committee on Cancer Criteria ((Hsueh et al;
2010)).
For immunohistochemical analyses, Nm23-H1 (Dako, USA),
Claudin 4 (Neomarker, LABVISION, USA), and Ki67 (Dako, USA),
prediluted ready to use, were applied for each case using the AvidinBiotin complex (APC) technique (Hsu et al., 1981). The procedure was
as follow: Slides were dewaxed in xylen, and then treated with
microwave heating at 90 C for 10 minutes in10 mmol/L citrate
monohydrate (PH 6.0) for antigen retrieval. One to two drops of each of
the primary monoclonal antibodies were applied to each section. Slides
were incubated in humid chamber for one hour at room temperature.
Then the sections were incubated for 20 min with a biotinylated
secondary solution, and then rinsed with distilled water. This was
followed by another 20 min of incubation with streptavidin solution.
Freshly prepared chromogen diaminobenzine (DAB) was incubated with
slides for 3-5 minutes then washed with distilled water. Slides were
counterstained for 3 minutes with Mayer's hematoxylin then washed and
covered with mounting media (DPX).
 Immunohistochemical Assessment:
Nm23-H1 is expressed in the cytoplasm of tumor cells as yellow
brown granules. A two point scale was used in its assessment as follows;
Negative for no significant tumor cell staining & Positive for cytoplasmic
staining of tumor cells (Xiang et al., 1998).
The expression of Claudin 4 was assessed by semiquantative scoring
of the extent and intensity of the staining. Only membranous staining was
classified as positive. The staining extent was represented by the
percentage of positively stained tumor cells and graded as less than 10%
(1+), in between 10-50% (2+), or more than 50% (3+).The staining
intensity was recorded as absent (0),weak (1+),moderate (2+), or strong
(3+).The 2 scores were multiplied to give a final score of 0 to 9,which is
grouped as low (final score 0-2) and high (final score 3-9) (Hsueh et
al.,2010)
MIB-1 immunoreactivity was assessed by counting the positive cells
that show nuclear staining for MIB-1, under x400 magnification fields.
Score for each tumor was reported as the means of the percentage of
positive cells per high power field (proliferation index) (Xu et al.,
2015).Using a cut off level of 20%, MIB-1 scores were ranked as high
proliferation index (HPI), if ≥20% of tumor cells were positive, and low
proliferation index (LPI), if <20% of tumor cells were positive (Guoqing
& Xiaoyan; 2004).
A negative control was used for each marker, by omitting the primary
antibody and replacing it with normal rabbit serum IgG.
 Statistical analyses:
All quantitative data are expressed as means ± standard deviation
(SD). Statistical Package for the Social Sciences (SPSS) program, version
16.0 for windows (SPSS, Inc., Chicago, IL, USA) was used, on a
personal computer. The Pearson correlation coefficient was used to
correlate Nm23-H1, Claudin-4 and MIB-1 expression with clinicopathological data. The Kaplan-Meier method was used to calculate
cumulative survival. Survival of patients was compared using the log
rank test to compare the survival curves.
 Results
Ages of the studied 50 cases of nasopharyngeal carcinoma ranged
from 22 to 76 years with mean age (45.3± 9.26). Out of 50 cases, 35
cases (70%) were males and 15 cases (30%) were females.
 Table (1): Demographic and clinico-pathological characteristics of
the 50 studied cases.
Characteristics
No. Of cases (%)
Total patients
50 (100%)
Age (Years)
<45
21 (40%)
>45
29 (60%)
Gender
Male
35 (70%)
Female
15 (30%)
T1
3 (6%)
T2
18 (36%)
T3
19 (38%)
T4
10 (20%)
N0
13 (26%)
N1
12 (24%)
N2
22 (44%)
N3
3 (6%)
M0
38 (76%)
M1
12 (24%)
I
2 (4%)
II
7 (14%)
III
18 (36%)
IV
23(46%)
Prognosis
5 year disease
10 (46%)
(Of 22 cases only)
free survival
Primary Tumor
Regional Lymph Nodes
Distant Metastasis
TNM Stage
Recurrence
4 (18%)
Death
8(36%)
 Immunohistochemical results:
Nm23-H1 Expression Results (Figures 1,2):
Staining of the product of Nm23-H1 gene was cytoplasmic.
Positive cytoplasmic expression was seen in 18 cases (36%); while 32
cases (64%) showed negative expression (Table 2). The nm23-H1
expression in relation to age and sex is shown in Table 2.
Nm23-H1 expression was insignificantly correlated with tumor
size (P value >0.05) (Table 2).
Nm23-H1 expression was significantly correlated to lymph node
status (P value <0.01) (Table 2).
Also the relation of nm23-H1 expression to distant metastasis was
statistically significant (P value <0.05) (Table 2).
Similarly the relation between nm23-H1 expression and TNM
stage was statistically significant (P value <0.01) (Table 2).
From previous results it was obvious that negative Nm23-H1
expression was associated with higher incidence of lymph node
metastasis, distant dissemination and advanced clinical stage than those
with positive Nm23-H1 expression.
Claudin-4 Expression Results (Figures 3,4):
Twenty cases (40%) showed high Claudin-4 expression, while 30
cases (60%) showed low expression for Claudin-4 (Table 2). The
Claudin-4 expression in relation to age and sex is shown in Table 2.
Claudin-4 expression was insignificantly correlated with tumor
extent (P value >0.05) (Table 2).
Similarly Claudin-4 expression was insignificantly correlated with
lymph node status (P value >0.05) (Table 2).
The relation of Claudin-4 expression to distant metastasis was
statistically significant (P value <0.01) (Table 2).
Also Claudin-4 expression was insignificantly correlated with
TNM stage (P value >0.05) (Table 2).
From previous results, it was found that low Claudin-4 expression
was associated with distant metastasis.
MIB-1 Expression Results (Figures 5,6):
Regarding MIB-1 expression; it ranked from 1- 40%; with a mean
of 16.08 ± 12.09, where 16 cases (32%) showed High Proliferation Index
for MIB-1 expression (HPI), while 34 cases (68%) showed Low
Proliferation Index (LPI) for MIB-1. The MIB-1 expression in relation to
age and sex is shown in Table 2.
MIB-1 expression was insignificantly correlated with tumor extent
(P value >0.05) (Table 2).
Similarly MIB-1 expression was insignificantly correlated with
lymph node status (P value >0.05) (Table 2).
The relation of MIB-1 expression to distant metastasis was
statistically insignificant (P value >0.05) (Table 2).
Also MIB-1 expression was insignificantly correlated with TNM
stage (P value >0.05) (Table 2).
Correlation of nm23-H1, MIB-1 and Claudin-4 Expression (Table 2):
Regarding the relation between the expression results of the three
used markers; the results were statistically insignificant, as out of 30
cases showing low expression for Claudin-4, 22 cases showed negative
expression and 8 cases showed positive expression for nm23-H1; while
20 cases expressed LPI and 10 cases expressed HPI for MIB-1
respectively; whereas out of 20 cases showing high expression for
Claudin-4, 10 cases showed negative expression and 10 cases showed
positive expression for nm23-H1; while 14 cases expressed LPI and 6
cases expressed HPI for MIB-1 respectively. Also out of 34 cases
showing LPI for MIB-1 expression; 20 cases were negative and 14 cases
were positive for nm23-H1; while out of 16 cases showing HPI for MIB1, 12 cases were negative and 4 cases were positive for nm23-H1.
Relation of Claudin-4, MIB-1 and nm23-H1 expressions to Prognosis
(Table 3):
Survival curves were plotted against nm23-H1, Claudin-4 and
MIB-1 expressions (Diagrams 1, 2 &3). Log rank test showed significant
correlations (P<0.05).
It was found that 15 out of 22 followed-up cases were negative for
nm23-H1; of which 3 cases recurred and 8 cases died; while 7 out of 22
cases were positive for nm23-H1; of which 6 cases showed disease free
survival for 5 years. Nm23-H1 expression indicated favorable prognosis;
with significant association (P value <0.05).
Also 13 out of 22 followed-up cases showed low Claudin-4
expression; of which 2 cases recurred and 7 cases died; whereas 9 out of
22 cases showed high Claudin-4 expression; of which 6 cases showed
disease free survival for 5 years. Low Claudin-4 expression correlated
significantly with shorter recurrence free survival, hence, unfavorable
prognosis (P value <0.05).
Regarding MIB-1 expression; 14 out of 22 followed-up cases
showed LPI for MIB-1; of which 8 cases showed disease free survival for
5 years; while 8 cases out of 22 followed-up cases showed HPI; of which
6 cases died. So; it can be suggested that High Proliferation Index was
associated with poorer prognosis than LPI; with significant association (P
value <0.05).
Table (2): Relation between nm23-H, Claudin-4, MIB-1 expressions &
clinico-pathological findings:
Nm23-H1
Clinicopathological
Data
-ve
+ve
(No.&%) (No.&%)
Claudin 4
P value
Low
High
(No.&%) (No.&%)
MIB-1
P value
LPI
HPI
P
(No.&%) (No.&%) value
Age
- <45
- >45
11 (34.4)
10 (55.6)
>0.05
10 (33.3)
11 (55)
20 (66.7)
9 (45)
23 (76.7)
12 (60)
>0.05
17 (50)
4 (25)
17 (50)
12 (75)
26 (76.5)
9 (56.3)
21 (65.6)
8(44.4)
- Male
23 (71.9)
12 (66.7)
- Female
9 (28.1)
6 (33.3)
7 (23.3)
8 (40)
8 (23.5)
7 (43.7)
- T1
1 (3.1)
2 (11.1)
3 (10)
0
2 (5.9)
1 (6.2)
- T2
12 (37.5)
6 (33.3)
12 (40)
6 (30)
15 (44.1)
3 (18.8)
- T3
11 (34.4)
8 (44.4)
10 (33.3)
9 (45)
13 (38.2)
6 (37.5)
- T4
8 (25)
2 (11.1)
5 (16.7)
5 (25)
4 (11.8)
6 (37.5)
- N0
2 (6.2)
11 (61.1)
5 (16.7)
8 (40)
8 (23.5)
5 (31.2)
- N1
11 (34.4)
1 (5.6)
9 (30)
3 (15)
12 (35.3)
0
- N2
16 (50)
6 (33.3)
13 (43.3)
9 (45)
13 (38.2)
9 (56.3)
- N3
3 (9.4)
0
3 (10)
0
1 (2.9)
2 (12.5)
- M0
21 (65.6)
17 (94.4)
19 (63.3)
19 (95)
26 (76.5)
12 (75)
- M1
11 (34.4)
1 (5.6)
11 (36.7)
1 (5)
8 (23.5)
4 (25)
-I
0
2 (11.1)
2 (6.6)
0
1 (2.9)
1 (6.2)
- II
4 (12.5)
3 (16.7)
3 (10)
4 (20)
6 (17.6)
1 (6.2)
- III
8 (25)
10 (55.5)
8 (26.7)
10 (50)
14 (41.2)
4 (25)
- IV
20 (62.5)
3 (16.7)
17 (56.7)
6 (30)
13 (38.2)
10 (62.5)
>0.05
Sex
>0.05
>0.05
>0.05
1ry Tumor
>0.05
>0.05
>0.05
LN
<0.01**
>0.05
>0.05
Distant
Metastasis
<0.05*
<0.01**
>0.05
TNM Stage
<0.01**
>0.05
>0.05
Nm23-H1
Clinicopathological
Data
-ve
+ve
(No.&%) (No.&%)
Claudin 4
P value
Low
High
(No.&%) (No.&%)
MIB-1
P value
LPI
HPI
P
(No.&%) (No.&%) value
NM23:
- Negative
22 (73.3)
10 (50)
8 (26.7)
10 (50)
>0.05
----
----
>0.05
20 (66.7)
10 (33.3)
14 (70)
6 (30)
30
20
-----
----
----
22 (68.8)
10 (31.2)
8 (44.4)
10 (55.6)
- High
20 (62.5)
12 (37.5)
14 (77.8)
4 (22.2)
Total
32
18
- Positive
Claudin 4:
- Low
- High
>0.05
20 (58.8)
12 (75)
>0.05
14 (41.2)
4 (25)
----
20 (58.8)
14 (41.2)
10 (62.5)
6 (37.5)
>0.05
>0.05
----
----
----
34
16
MIB-1:
- Low
N.B.: > 0.05= insignificant correlation, * <0.05= significant
correlation, ** <0.01= highly significant correlation
Table (3): Relation of nm23-H1, Claudin-4 and MIB-1 expressions to
clinical outcome:
Clinical
outcome
-Disease
Nm23-H1
-ve
+ve
(No.&%) (No.&%)
4 (26.7)
Claudin 4
P
value
6 (85.7)
Low
High
(No.&%) (No.&%)
4 (30.8)
MIB-1
P
value
6 (66.7)
LPI
HPI
(No.&%) (No.&%)
8 (57.1)
P
value
2 (25)
free
survival
-Recurrence
<0.05*
<0.05*
<0.05*
-Death
3 (20)
8 (53.3)
1 (14.3)
0
2 (15.4)
7 (53.8)
2 (22.2)
1 (11.1)
4 (28.6)
2 (14.3)
0
6 (75)
Total
15
7
13
9
14
8
Diagram (1): Relation of nm23-H1 expression to the Survival.
Diagram (2): Relation of claudin-4 expression to the Survival.
Diagram (3): Relation of MIB-1 expression to the Survival.
Figure (1) showing strong positive cytoplasmic expression for nm-23 in non-distant
metastasizing undifferentiated NPC (Immunostaining, x400).
Figure (2): showing negative cytoplasmic expression for nm-23 in distant
metastasizing undifferentiated NPC (Immunostaining, x400).
Figure (3): showing high Claudin-4 membranous expression in non-distant
metastasizing undifferentiated NPC (Immunostaining, x400).
Figure (4): showing low Claudin-4 membranous expression in distant metastasizing
undifferentiated NPC (Immunostaining, x400).
Figure (5): showing HPI for MIB-1 nuclear expression in distant metastasizing
undifferentiated NPC (Immunostaining, x400).
Figure (6): showing LPI for MIB-1 nuclear expression in non-distant metastasizing
undifferentiated NPC (Immunostaining, x400).
Discussion
Primary NPC has unique pathological and clinical characteristics
and radiotherapy with or without chemotherapy is the mainstream
treatment. Although the 5-year survival of patients with NPC has steadily
improved over the past three decades (Lee et al; 2005, Chan; 2011),
overall 15‑58% of patients experienced recurrence after radical
radiotherapy in the era of conventional radiotherapy (Chang et al; 2000),
and 13‑22% have experienced recurrence in the era of intensitymodulated radiation therapy (Ng et al; 2011, Su et al; 2011). In most
patients with recurrence after complete remission following radical
radiotherapy, the cancer returns within an average of 1.5 years, with local
recurrences accounting for 70% of such cases (Yang et al; 1996, Lee et
al; 1999). Recurrent NPC may be local, regional, or distant and is usually
treated with radiation therapy and/or chemotherapy and occasionally with
surgery. Retreatment for recurrent NPC poses a critical challenge given
its poor efficacy and serious toxicities (Xu et al; 2013). Improved
identification of prognostic factors by means of molecular testing may be
useful in the diagnosis of diseases and their exact subtypes and may aid
physicians in selecting individualized treatment, increasing the likelihood
of local salvage. Several prognostic factors have been identified in recent
years including recurrent tumor T stage, histologic type, patient age and
disease-free interval to recurrence (ITR). Of these factors, short-term ITR
has been shown to correlate with poor outcome (Chou et al; 2008, Xu et
al; 2015). Therefore, identification of molecular markers that may lead to
an improved understanding of recurrent NPC and to individualized
treatment is imperative.
It is generally accepted that decreased expression of claudins leads
to diminished cellular adhesion and differentiation with subsequent tumor
cell dissociation and invasion. The notion is supported by the previous
findings of reduced expression of claudins 1, 4, and 7 during progression
of high-grade intraepithelial neoplasia to invasive carcinomas in urinary
bladder (Boireau et al; 2007) and lost expression of claudins 1, 4, and 7
in diffuse type or poorly differentiated gastric adenocarcinoma (Resnick
et al; 2005, Johnson et al; 2005). It has been demonstrated that small
interfering RNA-mediated knockdown of claudin 7 in esophageal SCC
cell lines decreased E-cadherin expression and increased tumor cell
growth and invasion (Lioni et al; 2007).
However other studies revealed that overexpression of claudin 4
has been reported in carcinomas of breast, prostate, stomach, uterus,
ovary, pancreas, and kidney (Sato et al; 2004, Tokes et al; 2005, Resnick
et al; 2005, Zhu et al; 2006, Sheehan et al; 2007, Santin et al; 2007,
Lechpammer et al; 2008, Lanigan et al; 2009), with correlation to
aggressive tumor behavior or decreased survival. Also Lee et al (2009)
demonstrated that overexpression of claudin 1 confers resistance to cell
death in NPC cell lines.
In contrast, our study demonstrated the expression pattern of
claudin-4 in undifferentiated NPC patients and its association with
prognostic variables and survival and showed that there was a significant
correlation of high claudin-4 expression with lower rate of distant
metastasis and it was associated with improved distant metastasis free
survival (DMFS).
Consistent with our study results, it has been shown in pancreatic
cancer cell lines that up-regulation of claudin 4 diminished the
invasiveness and the metastatic potential of the cancer cells (Michl et al;
2003). This may be explained by the review held by Ding et al; 2013 who
reported that downregulation of claudins contributes to epithelial
transformation by increasing the paracellular permeability of nutreints
and growth factors to cancerous cells.
Possible explanation for studies revealing opposite opinion is
through the interaction with Zona Occludens-1 (ZO-1) involved in tight
junctions, to affect other cell signaling pathways involved in neoplastic
transformation (Resnick et al; 2005). Few other plausible mechanisms
have been proposed, one is through the suppression of apoptosis by
increased expression of claudin 1 in NPC cell lines (Lee et al; 2009) and
the other is through the activation of matrix metalloproteinase (MMP)
proteins (Oku et al; 2006, Agarwal et al; 2005). Up-regulation of claudin
1 in oral SCC enhances invasion by the activation of MMP-2 and MMP1, and overexpression of claudins 3 and 4 in ovarian surface epithelial cell
promotes invasion by increasing MMP-2 activity (Agarwal et al; 2005).
It is recognized that claudins 3 and 4 can function as receptors for
the Clostridium perfringens enterotoxin (CPE). CPE can cause cytolysis
upon binding to its receptors through the effects on membrane
permeability (Morin et al; 2005, Swisshelm et al; 2005, Santin et al;
2007). Therefore, cancer cells expressing claudins 3 and 4 can be the
potential target for CPE toxin mediated therapy. Experiments have
established that breast, ovarian, prostatic, and pancreatic cancer cells are
sensitive to CPE treatment (Morin et al; 2005). NPC may also be a
possible candidate for such therapy because positive immunoreactivity
for claudin-4 was detected in 60 % of undifferentiated NPC patients in
this study.
The nm23 gene is one of the tumor suppressor genes. The
expression of nm23-H1 has been reported to be inversely associated with
metastatic potentiality in a number of human carcinomas, including
breast, colorectal, gastric, hepatocellular and gall bladder carcinomas.
Also exogenous overexpression of the metastasis suppressor gene Nm23H1 reduces the metastatic potential of multiple types of cancer cells and
suppresses in vitro tumor cell motility and invasion (Xiang et al; 1998,
Horak et al; 2007).
This study revealed that reduced expression of nm23-H1 correlated
with node and distant metastasis, advanced TNM stage, recurrence and
poor survival and the results were statistically significant.
In agreement with the results of this study, Liu et al (2008)
reported that downregulation of nm23-H1 expression was associated with
intracranial invasion in NPC and hence poor prognosis. Also Xuebing et
al (2012) revealed that nm23-H1 downregulation is a biomarker for
metastasis and poor prognosis in NPC.
However, these findings cannot be generalized, and the biological
functions of nm23 in cancer cells are perplexed. A positive correlation
between nm23-H1 level and metastatic potential has also been observed
in other human tumors including pancreatic carcinoma (Nakamori et al.
1993), osteosarcoma (Oda et al; 2000), neuroblastoma (Almegren et al;
2004), and esophageal squamous cell carcinoma (Wang et al; 2004).
Given the complexity of metastasis, multiple pathways downstream of
nm23-H1 presumably mediate its phenotypic effects.
MIB-1 (Ki-67) is a maker of cell proliferation and has been widely
used to assess cell proliferation in neoplastic tissues. Many studies
indicate that MIB-1 has a close relationship with the replication,
progression and prognosis of tumors (Guoqing & Xiaoyan; 2004). In this
study, we detected the expression of MIB-1 in undifferentiated NPC in
order to study the relationship between biological behavior and prognosis
of NPC.
Although this study revealed insignificant results with TNM stage,
yet it revealed that HPI for MIB-1 protein was associated with poor
survival. Small number of cases tested may contribute to this
insignificance as regards TNM staging.
In contrast to our results Masuda et al (1998) found no association
with distant metastasis and survival and also zel et al (2004) reported LPI
of Ki67 in undifferentiated NPC.
Also Guoqing & Xiaoyan (2004) found that of 56 cases studied, 16
cases (28.6%) showed MIB-1 HPI and 40 cases (71.4%) showed MIB-1
LPI and also Xiaoyan et al (2005) reported that the patients having low
expression of ki67 were not sensitive to chemotherapy, liable to
metastasis to distant organs and had a poor prognosis, while tumors
which highly expressed Ki67 (high proliferated tumors) were sensitive to
radiotherapy and had a better prognosis.
In our study, the percentage of Ki67 positive cells in NPC was up
to 40% and the HPI accounted for 32% which was coincidence with the
studies in the literature (Gabusi et al; 2001, Xiaoyan et al; 2005). Our
statistical outcome indicated that the expression of Ki67 had no
relationship to the stage of NPC (P<0.05), but significantly correlated
with prognosis. A possible explanation could be that highly proliferating
tumors confers an aggressive biological behavior.
In agreement with our results, Raybaud et al (1997) found that
patients (with poorly differentiated squamous cell carcinoma, in head and
neck) who lowly expressed Ki67 would receive a better curative effect on
radiotherapy, even if they had a late clinical stage.
Meanwhile, according to the expression of MIB-1 before the
treatment, an optimal method could be chosen and a series of measures
can be taken to increase the cure rate of NPC patients. For example, we
may add new remedy that can increase the radiosensitivity to the patients
or use the adjuvant chemical treatment before or after the radiotherapy if
the patients were highly expressed MIB-1 and were not sensitive to the
radiotherapy.
In conclusion, Claudin-4 may serve as a useful biomarker for the
prediction of distant metastasis and patient survival in NPC. However,
more cases need to be studied to confirm these findings. Further
investigations are warranted to elucidate the role of claudins-4 in the
pathogenesis of NPC especially undifferentiated type for the development
of effective treatment. All the results of the used markers in relation to
prognosis were statistically significant in a meaning of low Claudin-4
expression; Negative nm23-H1 expression and HPI for MIB-1 were
associated with poor survival.
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