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Expressions of Neutrophil Gelatinase-Associated Lipocalin in Gastric CancerA Potential Biomarker for Prognosis and an Ancillary Diagnostic Test.

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THE ANATOMICAL RECORD 293:1855–1863 (2010)
Expressions of Neutrophil GelatinaseAssociated Lipocalin in Gastric Cancer:
A Potential Biomarker for Prognosis and
an Ancillary Diagnostic Test
HUI-JU WANG,1 XU-JUN HE,1 YING-YU MA,1 XIAO-TING JIANG,2 YING-JIE XIA,1
ZAI-YUAN YE,1 ZHONG-SHENG ZHAO,3 AND HOU-QUAN TAO1*
1
Key Laboratory of Gastroenterology of Zhejiang Province, Hangzhou, China
2
Clinical Laboratory, Zhejiang Provincial People’s Hospital, Hangzhou, China
3
Department of Pathology, Zhejiang Provincial People’s Hospital, Hangzhou, China
ABSTRACT
The aim of this study was to explore the clinical significance of neutrophil gelatinase-associated lipocalin (NGAL) in the development and prognosis of gastric cancer. NGAL tumor levels were determined in 333 GC
patients by immunohistochemistry. NGAL in blood samples from 63
healthy donors and 60 gastric cancer patients were also determined by
enzyme-linked immunosorbent assay. Rate of NGAL expression was correlated with the size of tumor (69.3% in >4 cm tumors vs. 46.1% in 4 cm
tumors), Lauren’s classification (84.3% in diffuse type vs. 28.2% in intestinal type), lymph node metastasis (75.6% vs. 16.4% with no metastasis),
vascular invasion (74.9% vs. 26.8% with no invasion), distant metastasis
(94.3% vs. 50.3% with no distant metastasis), and TNM stage (81.8% in
TNM IIIþIV vs. 20.5% in TNM IþII). NGAL expression can be used as an
independent prognostic factor in gastric cancer as indicated by multivariate analysis. Positivity for serum NGAL was higher than that for
carbohydrate antigen determinant, CA19-9 (38.1% vs. 12.5%) in TNM I,
and higher than that for carcinoembryonic antigen, CEA (58.3% vs. 8.3%)
and CA19-9 (58.3% vs. 8.3%) in TNM II. In conclusion, serum NGAL has
great potential to be used as an ancillary test for diagnosis of gastric
cancer. Increased expression of NGAL in tumors suggests gastric cancer is
likely to be at an advanced stage with invasion and metastasis, and also poor
C 2010 Wiley-Liss, Inc.
prognosis. Anat Rec, 293:1855–1863, 2010. V
Key words: NGAL; gastric cancer; neoplasm
neoplasm metastasis; diagnosis
INTRODUCTION
Gastric cancer is one of the most common cancers of
the digestive system and its incidence varies significantly in different countries and regions. China is one of
the country with a high number of gastric cancer cases
(third in the world) (Pavlick et al., 1997) and the main
causes appear to be related to environmental, genetic
and dietary factors. Based on a survey in the year 2000
(Parkin et al., 2001), the mortality attributed to gastric
cancer was 734,000 deaths annually worldwide, which
was the second most common cause of cancer mortality,
and the incidence of gastric cancer has been increasing
year on year. Biomarkers have been widely used for
C 2010 WILEY-LISS, INC.
V
invasiveness;
Additional supporting information can found in the online
version of this article.
Grant sponsor: Zhejiang Province Nature Science Foundation
of China; Grant number: Y207763; Grant sponsor: Zhejiang
Provincial Program for the Cultivation of High-level Innovative
Health Talents.
*Correspondence to: Hou-Quan Tao, Key Laboratory of Gastroenterology of Zhejiang Province, Hangzhou 310014, Zhejiang, China.
Fax: 0086-571-85131448. E-mail: taohouquan2008@yahoo.com.cn
Hui-Ju Wang, Xu-Jun He, and Ying-Yu Ma contributed equally to
this work.
Received 24 February 2010; Accepted 9 June 2010
DOI 10.1002/ar.21230
Published online 20 August 2010 in Wiley Online Library
(wileyonlinelibrary.com).
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WANG ET AL.
screening and early detection of gastric cancer for many
years, and can be relatively specific such as serum prostate-specific antigen for prostatic cancer (Brawer et al.,
1993; Catalona et al., 1993), serum carcinoembryonic
antigen (CEA) with high specificity but insufficient sensitivity for detection of colorectal cancer (CRC) recurrence
in isolation (Tan et al., 2009), and carbohydrate antigen
determinant 19-9 (CA19-9) for pancreatic/bile duct
cancers (Wu et al., 2006). However, there is no adequate
marker that has been developed and proven to be useful
clinically for diagnosis and prognosis of gastric cancer and
the lack of such biomarker(s) may have contributed to the
high mortality of gastric cancer in all cancer cases. As in
other cancers, the stage of the tumor (depth of invasion
and metastasis) is the key factor in prognosis and often
determines the overall survival rate. The process of tumor
invasion and metastasis involves multiple steps, although
these are not yet fully understood, including tumor cell
adhesion, interaction with the surrounding matrix and
migration. A biomarker that is biologically associated
with the process and is linked functionally with later
cancer stage would allow us to predict high risk of invasion and metastasis, and would be of great value.
Neutrophil gelatinase-associated lipocalin (NGAL), a
member of the lipocalin family, was originally identified
as a protein stored in specific granules of human neutrophils (Kjeldsen et al., 1993). It was observed to form a
complex with the gelatinase, matrix metalloproteinase-9
(MMP-9), and is composed of 178 amino acids (Kjeldsen
et al., 1993). It belongs to the lipocalin family (Flower,
1996) which comprises more than 50 known members,
all of which are characterized by their low molecular
weight and their ability to bind to and transport a group
of small lipophilic substances. NGAL exists as a 25 kDa
monomer, a 46 kDa disulfide-linked homodimer, and a
135 kDa disulfide-linked heterodimer with neutrophil
gelatinase (Kjeldsen et al., 1994). A cell type-specific
pattern of expression was seen in bronchus, stomach,
small intestine, pancreas, kidney, prostate gland, and
thymus (Friedl et al., 1999). In recent years, NGAL was
found abnormal expression in many malignant tumors
such as esophageal cancer (Zhang et al., 2007), ovarian
cancer (Lim et al., 2007), and pancreatic cancer (Furutani et al., 1998). Because its expression was often associated with a later stage of cancer, a role in tumor
invasion and metastasis was, therefore, proposed, and it
was also considered an independent indicator of poor
prognosis for some of the tumors (Bauer et al., 2008).
The characteristics of NGAL expression in gastric
cancer are still largely unknown. In this article, we studied neoplasm expression of NGAL in 333 cases of gastric
cancers using immunohistochemical staining, serum
levels of NGAL in 60 patients using sandwich enzymelinked immunosorbent assay (ELISA) and NGAL expression in five gastric cancer cell lines using reverse transcription-polymerase chain reaction (RT-PCR) and Western
blot analysis. The correlation between NGAL expression
and tumor invasion and metastasis, and the significance of
serum NGAL levels were analyzed.
tiation), SGC-7901 (moderate differentiation), BGC-823
(poor differentiation), AGS (poor differentiation), KatoIII (poor differentiation) were kindly provided by the
Digestive Surgery Institution, Ruijin Hospital of
Shanghai (Shanghai, China). They were cultured in
RPMI 1640 supplemented with 10% fetal bovine serum
and antibiotics (100 U/mL streptomycin and 100 U/mL
penicillin) and maintained at 37 C with 5% CO2. Cells
were passaged at 80% confluency using 1 mmol/L EDTA0.025% trypsin for 3–5 minutes.
Tissue Samples
Paraffinized specimens of tumors from 333 gastric
cancer patients who underwent curative gastrectomy
between 1995 and 2003 at the Department of Surgery
(Zhejiang Provincial People’s Hospital, Hangzhou,
China) were included. Written informed consent for
experimental immunohistochemistry was obtained from
all patients before analysis. The gastric cancer patients
were aged between 17 and 80 years old and had not
received radiotherapy or chemotherapy before surgery.
All of the cases were classified according to the WHO
Pathological Classification of Tumors, including 7 cases
of high differentiation, 101 cases of moderate differentiation, and 225 cases of poor differentiation. Fourteen
cases of papillary adenocarcinoma, 248 cases of tubular
adenocarcinoma, 23 cases of mucinous adenocarcinoma,
and 48 cases of signet-ring cell carcinoma according to
the histological classification. One-hundred seventy-four
cases of intestinal type and 159 cases of diffuse type
according to the Lauren classification. Two-hundred
seventeen cases with lymph node metastasis and
116 cases without lymph node metastasis. Seventy-three
cases at TNM stage I, 73 cases at TNM stage II, 138
cases at TNM stage III, and 49 cases at TNM stage IV.
Full follow-up data over 5 years, until December 2008,
were recorded for all patients.
In addition, 53 non-neoplastic paraffinized specimens
from normal gastric mucosa (n ¼ 26), gastritis (n ¼ 8),
and dysplasia (n ¼ 10) obtained by endoscopic biopsies,
and gastric ulcer (n ¼ 9) obtained by surgery, were
examined after obtaining appropriate consent.
Blood Samples and Isolation of Serum
Blood samples from 60 among the 333 gastric cancer
patients above (42 males and 18 females, mean age 56.1
years, range, 27–70) were also obtained. Sera from
63 healthy donors (35 males and 28 females, mean age
41.9 years, range, 25–65) were collected from October
2007 to February 2008. Whole blood was transferred
from an anticoagulant citrate dextrose solution formula
A-capped tube to a conical tube. Samples were centrifuged for 10 min at 1,500g in a swing bucket rotor at
4 C, and the serum was stored in cryovials at 80 C.
The project was approved by the ethics committee of
Zhejiang Provincial People’s Hospital.
RNA Isolation and RT-PCR Analysis
MATERIALS AND METHODS
Cell Lines
Five gastric adenocarcinoma cell lines at different
levels of differentiation including MKN45 (poor differen-
Total RNA from cell lines was isolated by Trizol (Invitrogen). A total of 2 lg RNA was reverse-transcribed
using the SuperScript II RNase-Reverse Transcriptase
System (Invitrogen). The cDNA was then subjected to
NGAL IN GC DIAGNOSIS AND PROGNOSIS
PCR with 1.5 mM magnesium chloride, 2.5U Taq polymerase in a total volume of 25 lL and specific primers
for NGAL. After 4 min initial denaturation at 94 C, 30
cycles of amplification (94 C for 30 s, 51 C for 30 s, 72 C
for 30 s) were performed. Beta-actin was used as an
internal control. The PCR products were electrophoretically resolved on 1% agarose gels stained with ethidium
bromide. Photographs were taken under ultraviolet
light, using the FluorChem software system (Alpha
Innotech). The primers of the forward and reverse for
NGAL (accession number: NM_005564.3) are 50 -GCA
CCA ACT ACA ACC AGC AT-30 and 50 -TTG GGA CAG
GGA AGA CGA T-30 (Invitrogen), respectively. The DNA
markers were bought from Beijing Dingguo Biotechnology Company, China.
1857
(provided by the manufacturer), sample (gastric cancer,
or healthy control serum sample), or sample diluent
alone (blank) was rapidly added to a 96-well plate precoated with NGAL antibody and incubated in 37 C for
120 min. Thereafter, 100 lL biotin-antibody working
solution was added to each well and incubated in 37 C
for 60 min and 100 lL horseradish peroxidase-avidin
working solution was added to each well. After incubated at 37 C for 60 min, the contents of the microwell
were aspirated and replaced with 90 lL of the chromogenic peroxidase substrate tetramethylbenzidine. The
plate was incubated for 30 min at 37 C (protected from
light), then the reaction was stopped by adding 50 lL
‘‘stop solution,’’ and the absorbance at 450 nm was read
with a microplate reader.
Cell Lysates and Western Blot
Cells were lysed in modified RIPA buffer (50 mM TrisHCl, pH 7.4, 0.25% Na deoxycholate, 150 mM NaCl, 1%
NP-40, 1 mM EDTA). Total cellular proteins were
extracted on ice for 30 min and centrifugated at 16,000g
for 10 min. The supernatant was collected and stored at
80 C until further use.
Samples were run on 10% sodium dodecyl sulfate-polyacrylamide gels and transfered onto polyvinylidene difluoride membranes. The membranes were blocked in 5%
bovine serum albumin and subsequently exposed to
primary antibodies specific for NGAL (rat monoclonal
anti-NGAL antibody, R&D systems, 1 lg/lL in PBS).
After incubation with the appropriate secondary antibody, the membranes were treated with ECL reagent
(Generay, China) and exposed to autoradiographic films.
Beta-actin was also detected as an internal control.
Immunohistochemical Staining
Briefly, each tissue section was deparaffinized, rehydrated and then incubated with fresh 3% hydrogen peroxide for 10 min. After rinsing with PBS, high pressure
antigen retrieval from the tissue was carried out in
0.01 M citrate buffer (pH 6.0). Next, sections were incubated with 10% normal goat serum for 15 min at room
temperature. After rinsing with PBS, slides were incubated with rat anti-human NGAL monoclonal antibody
(1:60 dilution in PBS, R&D Systems) overnight at 4 C.
After rinsing with PBS, tissue sections were incubated
for 20 min at room temperature with biotin-labeled
secondary antibody. After rinsing with PBS, tissue sections were incubated for 20 min at room temperature
with horseradish peroxidase polymer conjugate (Zymed).
Subsequently, they were stained with 3,3-diaminobenzidine, counterstained with hematoxylin, dehydrated and
mounted. Meanwhile, primary antibody replaced by PBS
was used as negative control.
Determination of Serum NGAL Levels by
Sandwich ELISA
The concentration of NGAL in the serum of patients
was determined using the NGAL ELISA Kit (Cusabio
Biotech). Serum samples from 60 patients with gastric
cancer were tested. Sera from 63 healthy donors were
used as control. A volume of 100 lL each of the standard
Measurement of Serum CEA and CA19-9
CEA and CA19-9 were measured with a commercially
available automated immunoassay method (Modular
Analytics, Roche Diagnostics) according to the manufacturer’s instructions. The upper limits of normal for this
method are 5.0 ng/mL for CEA and 37 U/mL for CA19-9.
Statistical Analysis
All slides were observed under a Nikon Light Microscope and representative photographs were taken. The
intensity of immunoreactivity of NGAL was scored. The
staining intensity was graded on a scale of 0 to 3þ (0
for no staining, 1þ for weak immunoreactivity, 2þ for
moderate immunoreactivity, and 3þ for strong immunoreactivity). The percentage of cells that showed positive
NGAL staining within the normal/cancerous region of a
section was scored as follows: 1: 0%–25% of cells positive, 2: 26%–50% positive, 3: 51%–75% positive, and 4:
76%–100% positive for NGAL. The staining intensity
score and the percent immunoreactivity score were
then multiplied to obtain a composite score. The values
of the composite score ranged from a minimum of 0 to a
maximum of 12, and 0 to 3 was defined as negative, 4
was defined as positive.
Statistical analyses of the data were performed with
SPSS V13.0 software. With regard to immunohistochemistry results provided by categorical scoring system,
Mann-Whitney U test was employed to compare two
groups of unpaired values, whereas for comparisons
between groups of more than two unpaired values,
we performed the Kruskal-Wallis H-test. The two independent sample t test was used to compare the mean serum NGAL value between normal and gastric cancer
samples. Correlations between TNM stage and serum
NGAL, CEA, CA19-9 were estimated by McNemar
paired-sample v2 test. The correlation of NGAL expression between serum and tissue was reported as Spearman’s product–moment correlation coefficients (rs).
Survival curves were constructed using the method of
Kaplan and Meier, including the Log-rank test. The Cox
proportional hazards model for multivariate survival
analysis was used to assess predictors related to
survival. All statistical tests were two-tailed, differences
were considered significant when P < 0.05.
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WANG ET AL.
RESULTS
NGAL Expression in Gastric Cancer Cell Lines
As shown in Fig. 1A, all five levels of differentiated
gastric cancer cell lines expressed NGAL. To confirm
this result, cell lysates from these cells were subjected to
Western blotting with anti-NGAL antibodies (Fig. 1B).
Consistent with the RT-PCR results, MKN-45, SGC7901, BGC-823, and AGS cells had high levels of NGAL
protein expression. However, the expression of NGAL
was dramatically lower in Kato-III cells than in any
other cells.
Immunohistochemical Staining Analysis
Fig. 1. (A) Reverse transcription-polymerase chain reaction for
neutrophil gelatinase-associated lipocalin (NGAL) expression by using
NGAL-specific primers with beta-actin as an internal control. (B)
NGAL expression was determined by Western blot analyses of protein lysates from gastric cancer cells with beta-actin as an internal
control.
Fig. 2. Results of NGAL immunohistochemistry, original magnification 200, the insert panes show the sections indicated by the arrows
at a magnification of 400. (A) Hematoxylin-eosin (HE) staining in
poorly-differentiated adenocarcinoma. (B) Immunostaining of NGAL
(yellow-brown granules, mainly in the cytoplasm) in poorly-differentiated adenocarcinoma. (C) HE staining in moderately-differentiated
adenocarcinoma. (D) Immunostaining of NGAL in moderately-differentiated adenocarcinoma. (E) HE staining at the edge of the adenocarcinoma with tumor invasion. (F) Immunostaining of NGAL at the edge of
the adenocarcinoma with tumor invasion. (G) HE staining in a lymph
Specimens from a total of 333 gastric cancer patients
with positive staining for NGAL showed cytoplasmic
yellow-brown granules (Fig. 2B,D) with appropriate
negative control (Fig. 2L, PBS replacing primary antibody against NGAL). The NGAL-positive detection rate
was 55.0% (183/333) in the gastric carcinoma specimens,
and the NGAL detection rate was statistically correlated
with the tumor size, Lauren classification, lymph node
node with metastasis in poorly-differentiated gastric adenocarcinoma.
(H) Immunostaining of NGAL in a lymph node with metastasis in
poorly-differentiated gastric adenocarcinoma. (I) HE staining in a
lymph node with metastasis in moderately-differentiated gastric adenocarcinoma. (J) Immunostaining of NGAL in a lymph node with metastasis in moderately-differentiated gastric adenocarcinoma. (K) HE
staining in gastric adenocarcinoma. (L) Negative control for immunostaining of NGAL, with phosphate-buffered saline replacing primary
antibody against NGAL.
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NGAL IN GC DIAGNOSIS AND PROGNOSIS
TABLE 1. Correlation of NGAL expression and
clinicopathological features of 333 gastric cancers
NGAL expression
Characteristics
Sex
Male
Female
Tumor diameter
4 cm
>4 cm
Lauren classification
Diffuse type
Intestinal type
Differentiation
Well
Moderate
Poor
Histology type
Papillary adenocarcinoma
Tubular adenocarcinoma
Mucinous adenocarcinoma
Signet-ring cell carcinoma
Lymph node metastasis
No
Yes
Vascular invasion
No
Yes
Distance metastasis
No
Yes
TNM stage
I þ II
III þ IV
Negative
Positive
P
value
116
34
121 (51.1%)
62 (64.6%) 0.025
111
39
95 (46.1%)
88 (69.3%) 0.000
25
125
134 (84.3%)
49 (28.2%) 0.000
5
43
102
2 (28.6%)
58 (57.4%)
123 (54.7%) 0.329
6
115
6
23
8
133
17
25
(57.1%)
(53.6%)
(73.9%)
(52.1%) 0.295
97
53
19 (16.4%)
164 (75.6%) 0.000
101
49
37 (26.8%)
146 (74.9%) 0.000
148
2
150 (50.3%)
33 (94.3%) 0.000
116
34
30 (20.5%)
153 (81.8%) 0.000
metastasis, vascular invasion, distant metastasis, and
TNM stage (Table 1). The detection rate of NGAL was
69.3% (88/127) in gastric carcinoma specimens of tumor
size >4 cm, which was higher than that in specimens of
tumor size 4 cm (46.1%, 95/206, P < 0.001). The frequency of NGAL positivity in samples from patients
with diffuse histological type (84.3%, 134/159) was significantly higher than that in patients with intestinal
histological type (28.2%, 49/174, P < 0.001). NGAL was
detected in 75.6% (164/217) of gastric cancer specimens
with lymph node metastasis, which was higher than in
specimens without lymph node metastasis (16.4%, 19/
116, P < 0.001). The detection rates of NGAL were
74.9% (146/195) and 94.3% (33/35) in specimens with
vascular invasion and distant metastasis, which were
higher than in specimens without vascular invasion
(26.8%, 37/138, P < 0.001) or distant metastasis (50.3%,
150/298, P < 0.001). NGAL was detected in 20.5% (30/
146) of TNM stage IþII samples, which was lower than
in TNM stage IIIþIV samples where it was detected in
81.8% (153/187, P < 0.001). NGAL expression appeared
to be greater in infiltrating tumor cells bordering the adjacent normal tissue (Fig. 2F) and metastatic foci in the
lymph nodes were often positive (Fig. 2H,J), which was
consistent with the proposition that NGAL is involved in
tumor invasion and metastasis.
The mean survival time in patients positive for NGAL
was 35.58 1.16 months, which was significantly lower
than in patients negative for NGAL (54.37 0.71
months, P < 0.001). The 5-year survival rate in patients
Fig. 3. Kaplan-Meier survival curve of gastric cancer patients positive and negative for NGAL expression.
TABLE 2. Multivariate analysis as determined
by Cox regression analysis in 333 gastric
cancer patients
95%
Confidential
interval
Clinicopathological
parameters
Lower
Upper
Hazard
ratio
P
value
Lauren classification
Vascular invasion
TNM stage
NGAL expression
1.571
1.176
1.048
1.217
3.466
2.766
2.625
2.788
2.334
1.804
1.659
1.842
0.000
0.007
0.031
0.004
with NGAL expression was significantly lower (20.8%)
than in patients without NGAL expression (66%, P <
0.001) (Fig. 3). Cox multivariate analysis showed that
survival was independently correlated with Lauren classification, vascular invasion, TNM stage and NGAL
expression (Table 2).
To evaluate the profile of NGAL in the carcinogenesis
of gastric cancer, we separately characterized the pattern of expression of NGAL in non-neoplastic gastric
tissues. In normal gastric mucosa, NGAL staining was
observed in neutrophils which invaded glands of the
lamina propria in gastritis, however, no NGAL expression was detected in normal gastric epithelial cells.
In gastric ulcers, NGAL staining was only detected in
neutrophils of necrosis foci. All the dysplasia was positive for NGAL expression (Fig. 4). This suggested that
the expression of NGAL was up-regulated in the early
stages of gastric carcinogenesis.
Quantitative ELISA for NGAL Levels in Serum
The serum levels of NGAL in normal versus gastric
cancer samples were analyzed by the two independent
sample t test. It showed that the NGAL level was high
in gastric cancer patients, but very low in healthy
donors (P < 0.001, Supporting Information Table 1). A
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WANG ET AL.
Fig. 4. NGAL staining analysis in tissue of normal and gastric dysplasia. (A) HE staining in normal gastric tissue. (B) NGAL expression
in normal tissue was negative. (C) HE staining in tissue of chronic gastritis in the active phase. (D) NGAL staining was detected in neutrophilic granulocytes when they invaded lamina propria glands in
chronic gastritis in the active phase. (E) HE staining in gastric ulcer.
(F) NGAL positive staining was detected in neutrophilic granulocytes
of necrosis foci in the gastric ulcer, but was negative in any other
location. (G) HE staining in tissue of gastric dysplasia. (H) NGAL
expression was positive in tissue of gastric dysplasia.
receiver operating characteristic (ROC) curve analysis
revealed that the area under the curve was 0.927,
suggesting that the test was fairly accurate in classifying cases as ‘cancer’ or ‘‘noncancer’’ (Fig. 5).
Relation of NGAL Expression Between
Serum and Tissue
When considering the relationship of NGAL expression between serum and tissue, a direct correlation was
observed between serum NGAL levels and NGAL
expression in gastric cancer. Twenty of the 26 cases with
an increased level of serum NGAL showed strong positive immunostaining of tumor cells, while only 13 of the
34 cases with normal serum levels of NGAL exhibited
positivity (R ¼ 0.385, P ¼ 0.002, Spearman’s q-test).
Relation Between Serum Concentrations of
NGAL, CEA, CA19-9, and TNM Stage
Simultaneously, we analyzed serum levels of CEA and
CA19-9 in the 60 gastric caner patients, and the data of
serum NGAL, CEA, and CA19-9 were listed in Supporting Information Table 2. Using an established cutoff for
healthy individuals of 14.31 ng/mL (the mean level þ 2
standard deviations in healthy controls), the detection
rate of serum NGAL reached 43.3% in the gastric cancer
samples. In contrast to serum levels of CEA and CA19-9
(CEA level >5 ng/mL and CA19-9 level >37 U/mL were
considered positive), the positive rate of serum NGAL
(38.1%) was significantly higher than serum CA19-9
(12.5%, P ¼ 0.016) in TNM stage I, and was significantly
higher than serum CEA (58.3% vs. 8.3%, P ¼ 0.031) and
CA19-9 (58.3% vs. 8.3%, P ¼ 0.031) in TNM stage II
(Table 3).
DISCUSSION
Proteins secreted by malignant cells that degrade the
extracellular matrix often play a key role in tumor inva-
Fig. 5. Receiver operating characteristic (ROC) curve analysis of
diagnostic sensitivity and specificity of the serum NGAL enzymelinked immunosorbent assay. The performance of the assay in discriminating patients with gastric cancer from normal subjects was
evaluated. The area under the curve was 0.927, suggesting that the
test was fairly accurate in distinguishing between the two groups.
sion and metastasis. MMP is one of the most important
proteins associated with tumor invasion and metastasis,
and can not only degrade the basement membrane and
matrix to promote tumor invasion and metastasis, but
also promote tumor growth and proliferation by angiogenesis. NGAL was originally found as a protein stored
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NGAL IN GC DIAGNOSIS AND PROGNOSIS
TABLE 3. Correlation between TNM stage and serum NGAL, CEA, CA19–9 in 60 gastric cancers
TNM
stage
I
II
III
IV
Rate of expression
positive for NGAL
38.1%
58.3%
41.2%
40.0%
(8/21)
(7/12)
(7/17)
(4/10)
Rate of expression
positive for CEA
14.3%
8.3%
17.6%
50.0%
(3/21)
(1/12)
(3/17)
(5/10)
Rate of expression
positive for CA199
12.5%
8.3%
23.5%
10.0%
(1/21)
(1/12)
(4/17)
(1/10)
P1 value
P2 value
0.227
0.031
0.289
1.000
0.016
0.031
0.508
0.375
Using an established cutoff for healthy individuals (14.31 ng/ml), 14.31 ng/ml was positive for NGAL; and 5 ng/ml was
positive for CEA, 37 U/ml was positive for CA19–9; P1: NGAL versus CEA; P2: NGAL versus CA19–9.
in specific granules of human neutrophils (Elneihoum
et al., 1996), and can bind and mediate the activity of
MMP-9 (Yan et al., 2001). It can also bind bacterial siderophores, preventing bacteria from retrieving iron from
this source (Goetz et al., 2002; Yang et al., 2002). Elevated NGAL expression was also observed in human
cancers such as colorectal, breast, pancreatic and ovarian cancers (Furutani et al., 1998; Friedl et al., 1999;
Lim et al., 2007; Zhang et al., 2007), may participate in
tumor invasion and is associated with poor differentiation of cancer cells (Kubben et al., 2007; Bauer et al.,
2008).
NGAL could act as a biomarker for the early stages of
pancreatic cancer, as evidenced by the fact that well to
moderately differentiated pancreatic cancer cells had
very high levels of NGAL expression, whereas NGAL
expression in poorly differentiated pancreatic cancer cell
lines was undetectable (Moniaux et al., 2008; Tong et al.,
2008). Interestingly, NGAL overexpression in this case
actually reduced pancreatic cancer cell adhesion and,
therefore, invasion in vitro and in vivo (Tong et al.,
2008). To support this idea, Lee et al. (2006) reported
that the overexpression of NGAL blocked human colon
cancer KM12SM cell invasion and liver metastasis. In
contrast, Li et al. (2003) reported the downregulation of
NGAL by antisense suppressed human esophageal carcinoma SHEEC cell invasion in vivo. These apparently
conflicting observations could be result from distinct
functions of NGAL in different cell types. Our current
study demonstrated that NGAL was expressed in most
of the gastric cancer cell lines, indicating that NGAL is
expressed universally in cancers originally from gastric
epithelium. No significant differences of NGAL expression were observed in MKN45, SGC-7901, BGC-823,
AGS and Kato-III cell lines. The results indicated that
NGAL expression was not correlated with the degree of
differentiation of gastric cancer.
The expression of NGAL has been demonstrated in
several other types of cancer, such as carcinoma of lung
(Friedl et al., 1999), gastric cancer (Kubben et al., 2007),
breast cancer (Stoesz et al., 1998), colon cancer (Nielsen
et al., 1996), pancreatic cancer (Argani et al., 2001; Han
et al., 2002), and ovarian cancer (Bartsch and Tschesche,
1995). However, no expression was detected in lymphoma and thymic carcinoma (Friedl et al., 1999). Our
study found that NGAL expression was significantly
higher in gastric cancer than that in normal tissue, suggesting that the expression of NGAL in cancer has tissue
specificity. Findings of research on esophageal squamous
cell carcinoma (ESCC) suggested that NGAL was
involved in the differentiation pathway and invasive progression of ESCC (Zhang et al., 2007), but another study
found that the NGAL expression was not only suppressing the ability of colon carcinoma cells to invade Matrigel in vitro, but also substantially inhibited liver
metastasis in an experimental animal model (Lee et al.,
2006). Our study found that the NGAL detection rate
was statistically correlated with tumor size, Lauren classification, lymph node metastasis, vascular invasion, distant metastasis, and TNM stage. The frequency of
positivity for NGAL in cases with tumor size >4 cm was
higher than in tumors 4 cm, and was higher in cases
with diffuse histological type (84.3%) than those with intestinal histological type (28.2%), and was also higher in
cases with lymph node metastasis (75.6%), vascular
invasion (74.9%), and distant metastasis (94.3%) and
TNM IIIþIV staging (81.8%) than in those without
lymph node metastasis (16.4%), vascular invasion
(26.8%) or distant metastasis (50.3%) and with TNM
IþII staging (20.5%). Therefore, NGAL may play an important role in invasion and metastasis of gastric cancer.
In recent years, the MMP family has been found to play
an important role in tumor invasion, metastasis and
recurrence (Takahashi et al., 2002; Masaki et al., 2003;
Curran et al., 2004). Collagen IV, which is a major component of basement membrane, also acts as a natural
barrier of cancer infiltration, and MMP-9 is one of the
IV collagenases. In vitro experiments suggested a possible role for NGAL in the protection of MMP-9 against
autolysis. In vivo data showed that enhanced levels of
MMP-9/NGAL complexes were significantly correlated
with the classifications of Lauren, and highly associated
with worse survival in gastric cancer (Kubben et al.,
2007). Although this article did not measure MMP-9 levels in samples, we also have reason to believe, in light of
Bubben’s previous work, that overexpression of NGAL in
gastric cancer may be associated with the activity of
MMP-9, thereby promoting the invasion and metastasis
of gastric cancer. NGAL overexpression was significantly
correlated with poor prognosis, suggesting that NGAL
may participate in the progression of gastric cancer, and
may be an indicator for poor prognosis of gastric cancer
patients. We also found that NGAL expression was negative in normal gastric glands, apart from neutrophils in
gastritis and gastric ulcer. However, all the gastric dysplasias were positive for NGAL expression. Our results
indicate that NGAL is upregulated in the early stage of
gastric carcinogenesis and may play an important role
in the early development of gastric cancer. The results
also suggest that NGAL could serve as an early diagnostic marker for gastric cancer especially in fine needle
aspirates or endoscopically-obtained biopsies.
Serum NGAL level has been shown to be elevated
early in acute renal injury (Mishra et al., 2005; Dent
et al., 2007) and ovarian borderline cancer (Lim et al.,
1862
WANG ET AL.
2007). The NGAL-MMP level was significantly higher in
the urine of breast cancer patients than in normal subjects, and the detection rate was nearly 84% (Fernandez
et al., 2005). Studies (Moniaux et al., 2008) have also
suggested that the serum NGAL level could be explored
as a possible diagnostic marker in pancreatic cancer in
the appropriate clinical context. Our research found that
serum NGAL levels in gastric cancer patients was
higher than that in healthy donors indicating that serum NGAL was associated with gastric cancer and can
be a significant index for diagnosis. To confirm that the
serum NGAL detected by our ELISA was derived from
tumor cells, we performed immunohistochemical analysis in all 60 gastric cancer specimens from patients
whose serum NGAL was tested. Summarizing the correlation between serum NGAL levels and NGAL expression in gastric cancer, 20 of the 26 cases with an
increased level of serum NGAL also showed strong positive immunostaining of tumor cells, while only 13 of the
34 cases with normal serum levels of NGAL exhibited
positivity. A significant correlation was observed
between serum NGAL level and NGAL expression in tumor cells (P < 0.05), suggesting that the serum NGAL
levels could be derived from tumor cells. Although serum
markers such as CA19-9 and CEA were highly elevated
in a few patients with advanced gastric cancer, these
conventional markers are usually not elevated in earlystage gastric cancer, and thus are not useful for detecting such cancers. ROC curve analysis revealed that the
area under the curve was 0.927 indicating our test was
fairly accurate in classifying cases as ‘cancer’ or ‘noncancer’. Since serum NGAL behaves in a different manner from CA19-9 or CEA, serum NGAL is likely to be
more effective than serum CA19-9 or CEA in distinguishing between patients with gastric cancer from
healthy controls. Notably, the present study showed that
the rate of positive serum NGAL expression was significantly higher than for serum CA19-9 in TNM stage I,
and was significantly higher than for serum CEA and
CA19-9 in TNM stage II. This suggests that serum
NGAL might have some potential to help detect early
gastric cancer. However, to clearly determine this, further studies analyzing a large number of patients with
early-stage gastric cancer are required.
In conclusion, measurement of serum NGAL has great
potential as an ancillary test for diagnosis of gastric cancer, and high expression of NGAL in gastric cancer tissues indicates tumor metastasis and poor prognosis.
Therefore, NGAL can be used as an indicator of invasion, metastasis and poor prognosis of gastric cancer.
The specific role of NGAL requires further elucidation in
future studies.
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expressions, test, associates, gelatinase, gastric, ancillary, lipocalin, prognosis, diagnostika, potential, neutrophils, cancer, biomarkers
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