The Prognostic Value of Tumor-Infiltrating Neutrophils in
Gastric Adenocarcinoma after Resection
Jing-jing Zhao1., Ke Pan1., Wei Wang1,2, Ju-gao Chen1, Yan-heng Wu4, Lin Lv1, Jian-jun Li1,
Yi-bing Chen1, Dan-dan Wang1, Qiu-zhong Pan1, Xiao-dong Li1,3*, Jian-chuan Xia1*
1State Key Laboratory of Oncology in Southern China and Department of Experimental Research, Sun Yat-sen University Cancer Center, Guangzhou, People’s Republic of
China, 2Department of Gastric and Pancreatic Surgery, Sun Yat-sen University Cancer Center, Guangzhou, People’s Republic of China, 3Department of Thoracic
Oncology, Sun Yat-sen University Cancer Center, Guangzhou, People’s Republic of China, 4First Affiliated Hospital of Jinan University, Jinan University, Guangzhou,
People’s Republic of China
Background: Several pieces of evidence indicate that tumor-infiltrating neutrophils (TINs) are correlated to tumor
progression. In the current study, we explore the relationship between TINs and clinicopathological features of gastric
adenocarcinoma patients. Furthermore, we investigated the prognostic value of TINs.
Patients and Methods: The study was comprised of two groups, training group (115 patients) and test group (97 patients).
Biomarkers (intratumoral CD15+ neutrophils) were assessed by immunohistochemistry. The relationship between
clinicopathological features and patient outcome were evaluated using Cox regression and Kaplan-Meier analysis.
Results: Immunohistochemical detection showed that the tumor-infiltrating neutrophils (TINs) in the training group ranged
from 0.00–115.70 cells/high-power microscopic field (HPF) and the median number was 21.60 cells/HPF. Based on the
median number, the patients were divided into high and low TINs groups. Chi-square test analysis revealed that the density
of CD15+ TINs was positively associated with lymph node metastasis (p=0.024), distance metastasis (p=0.004) and UICC
(International Union Against Cancer) staging (p=0.028). Kaplan-Meier analysis showed that patients with a lower density of
TINs had a better prognosis than patients with a higher density of TINs (p=0.002). Multivariate Cox’s analysis showed that
the density of CD15+ TINs was an independent prognostic factor for overall survival of gastric adenocarcinoma patients.
Using another 97 patients as a test group and basing on the median number of TINs (21.60 cells/HPF) coming from the
training group, Kaplan-Meier analysis also showed that patients with a lower density of TINs had a better prognosis than
patients with a higher density of TINs (p=0.032). The results verify that the number of CD15+ TINs can predict the survival
of gastric adenocarcinoma surgical patients.
Conclusions: The presence of CD15+ TINs is an independent and unfavorable factor in the prognosis of gastric
adenocarcinoma patients. Targeting CD15+ TINs may be a potential intervenient therapy in the future.
Citation: Zhao J-j, Pan K, Wang W, Chen J-g, Wu Y-h, et al. (2012) The Prognostic Value of Tumor-Infiltrating Neutrophils in Gastric Adenocarcinoma after
Resection. PLoS ONE 7(3): e33655. doi:10.1371/journal.pone.0033655
Editor: Irene Georgakoudi, Tufts University, United States of America
Received September 1, 2011; Accepted February 17, 2012; Published March 19, 2012
Copyright: ? 2012 Zhao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Natural Science Foundation of China (u0772002, 30700985, 30973398) and Guangdong Natural Science
Foundation (925100890). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (XdL); email@example.com (JcX)
. These authors contributed equally to this work.
Gastric carcinoma is one of the most common malignancies,
which account for a significant number of cancer-related deaths in
the world [1–2]. Gastric adenocarcinoma is the most common
type of gastric carcinoma. Despite the great advancement in
diagnosis and treatment modalities, especially surgical and
targeted therapies, the survival rate remains very low at a rate of
5 years . Increasing evidence suggests that inflammation is the
seventh hallmark of cancer . Leukocytes that infiltrated the
tumor promote tumor angiogenesis, growth and invasion [4–7].
The type, density, and location of tumor-infiltrating immune cells
in the local microenvironment have been linked to the clinical
outcome of various cancer types, including gastric cancer [5,8–10].
Yuan et al. showed that the elevated expression of Foxp3 in tumor-
infiltrating Treg cells suppressed T-cell proliferation and contrib-
uted to gastric cancer progression in a COX-2-dependent manner
. A study by Zhang et al. indicated that Th17 cells may
contribute to gastric cancer pathogenesis . Chen et al. also
demonstrated that the expression levels of IL-17 in the tumor were
an independent prognostic indicator in gastric adenocarcinoma
Neutrophils represent the 50%–70% fraction of total circulating
leukocytes [13–14]. Several pieces of evidence showed that
neutrophils promoted cancer cell migration and invasion  as
well as tumor-induced angiogenesis [16–18]. The presence of
PLoS ONE | www.plosone.org1 March 2012 | Volume 7 | Issue 3 | e33655
intratumoral neutrophils is a poor independent prognostic factor
in localized renal cell carcinoma . Because the role of CD15+
TINs in gastric adenocarcinoma has not been previously
elucidated, we investigated the level of CD15+ TINs by
immunohistochemistry and its relationship with clinicopatholog-
ical features in the current study. Furthermore, we evaluated its
prognostic value to post-resection survival in gastric adenocarci-
Immunohistochemical characteristics and association of
intratumoral CD15+ TINs with clinical and
histopathologic variables in the training group
Infiltrations of neutrophils were identified in the intratumoral
stroma. Our results show variation in the level of tumor infiltrating
neutrophils (TINs) (Fig. 1). In the training group which had115
cases, the number of CD15+ TINs ranged from 0.00–115.70 cells/
HPF, and the median number was 21.60 cells/HPF.
To investigate the association between the density of CD15+
TINs and the clinical features in gastric adenocarcinoma patients,
the median density of CD15+ TINs (21.60 cells/HPF) was used to
separate the patients in the training group into high and low TINs
groups (Table 1). Thus, the patients whose CD15+ TINs density
above 21.60 cells/HPF were defined as high level group, and the
rest were defined as low level group. The density of CD15+ TINs
was positively associated with lymph node metastasis (p=0.024),
distance metastasis (p=0.004) and UICC staging (p=0.028). No
correlations were found between the density of CD15+ TINs and
the tumor size (p=0.191), the depth of invasion (p=0.823), the
histologic grade (p=0.322), the gender (p=0.092) and the age
Correlation between CD15+ TINs and survival of patients
with gastric adenocarcinoma in the training group
The prognostic value of CD15+ TINs in the overall survival of
gastric adenocarcinoma patients was evaluated between patients
with a high or low density of infiltrating CD15+ neutrophils. The
high density of CD15+TINs was significantly associated with poor
prognosis of gastric adenocarcinoma patients using the Kaplan–
Meier curve analysis. Gastric adenocarcinoma patients with a high
density of CD15+ TINs had significantly lower overall survival
rates compared to the overall survival rate of patients with a low
density of CD15+ TINs (Fig. 2, p=0.002, long rank test).
Univariate and multivariate analyses of prognostic
variables in gastric adenocarcinoma patients
To identify the variables of potential prognostic significance in
the patients with gastric adenocarcinoma, univariate and multi-
variate analyses were further evaluated using the Cox proportion-
al-hazard model to compare the impact of CD15+ TINs and other
clinical pathological parameters on the prognosis of the 115
patients in the training group. The univariate analysis identified
that the CD15+ TINs (p=0.003), the tumor size (p=0.017), the
depth of invasion (p=0.010), the lymph node metastasis (p,0.001)
and the distance metastasis (p,0.001) were significantly associated
with overall survival (Table 2). Results from the multivariate
analysis showed that the CD15+ TINs (p=0.027), the depth of
invasion (p=0.041), the lymph node metastasis (p=0.015) and the
distance metastasis (p=0.040) were independent prognostic
factors of overall survival (Table 2). The relative risk in patients
with high levels of CD15+ TINs was 1.943 times greater than that
of patients with low levels of CD15+ TINs. Therefore, the level of
CD15+ TINs may predict the overall survival in patients with
Validation of CD15+ TINs for survival prediction in the
To validate CD15+TINs for prediction survival of gastric
adenocarcinoma patients, another 97 patients who underwent
surgical resection in the same hospital during 2002 to 2005 were
used as a test group. The 97 patients were divided into high
CD15+ TINs group (n=45, § 21.60 cells/HPF) and low CD15+
TINs group (n=52, ,21.60 cells/HPF) based on the median
density of CD15+ TINs (21.60 cells/HPF), which was obtained
from the training group. Kaplan-Meier survival analysis on the
Figure 1. The level of CD15+ + tumor-infiltrating neutrophils (TINs) in gastric adenocarcinoma surgical specimens shown by
immunohistochemistry. (A), (B), (E) and (F): High density of CD15+ TINs. (C), (D), (G) and (H): Low density of CD15+ TINs. Original
magnification: A–D6200; E–H6400.
Intratumoral Neutrophils in Gastric Adenocarcinoma
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patients with high CD15+ TINs infiltrating and low CD15+ TINs
infiltrating also was conducted as did in the training group. The
additional experiments results also showed that the patients with
the high density of CD15+ TINs were markedly poorer than those
of patients with the low density of CD15+ TINs. (Fig. 3, p=0.032,
long rank test).
In the current study, we performed the immunohistochemical
analysis to determine the CD15 expression, which is a marker of
neutrophils in gastric adenocarcinoma. We investigated the
interrelation of the level of CD15+ TINs and the clinicopatho-
logical parameters in gastric adenocarcinoma. The CD15+ TINs
were significantly associated with UICC staging (p=0.028), lymph
node metastasis (p=0.024) and distance metastasis (p=0.004)
(Table 1). Therefore, we believed that the CD15+ TINs are more
likely to contribute to tumor growth, progression and metastasis.
The mechanism has not been elucidated, and there are few
directed reports that address the mechanism. We hypothesize
TINs may promote the migratory activity of tumor cells. Carina et
al. have shown that neutrophil granulocytes induced intracellular
signaling in tumor cells through ICAM-1 clustering and promoted
the migratory activity of MDA-MB-468 human breast carcinoma
cells . Another possible mechanism is through angiogenesis.
Kuang et al. showed that high infiltration of peritumoral
neutrophils was positively correlated to angiogenic progression at
the tumor-invading edge in HCC patients. The researchers also
found that selective depletion of neutrophils effectively inhibited
tumor angiogenesis and growth in vivo . Infiltrating
neutrophils also played a crucial role in activating angiogenesis
in previously quiescent vasculature during the early stages of
carcinogenesis [7,21]. Although the results from the current study
show a correlation between the number of CD15+ TINs and
overall survival, the mechanism of TIN action to mediate survival
remains to be elucidated.
The Kaplan–Meier survival analysis revealed that the high level
of CD15+ TINs significantly correlated to the shorter survival time
of gastric adenocarcinoma patients (Fig. 2, p=0.002, long rank
test). The result of high level of CD15+ TINs significantly
associated with poor prognosis was also validated in the test group
(Fig. 3, p=0.032, long rank test). Furthermore, the univariate
analysis indicated that the level of CD15+ TINs was a significant
risk factor (HR=2.296, p=0.003) that affected the overall survival
of gastric adenocarcinoma patients. The multivariate analysis
demonstrated that the level of CD15+ TINs was an independent
predictor (HR=1.943, p=0.027) of overall survival (OS) in gastric
adenocarcinoma patients (Table 2). These results suggest that
CD15+ TINs may be a novel prognostic indicator for gastric
cancer. Coincident with our results, Hanne Krogh Jensen et al.
reported that the presence of intratumoral neutrophils was as a
poor independent prognostic factor for OS following nephrectomy
in localized clear cell RCC . Li et al. recently determined that
Table 1. Relationship between CD15+TINs and
clinicopathological features of gastric adenocarcinoma
patients in the training group.
All cases11557 58
,60 59 2930
Female 4116 25
,4 3219 13
§4 8338 45
Depthof invasion 0.823
UICC staging 0.028a
I–II43 27 16
III–IV 72 3042
N1–N3 7833 45
Distance metastasis 0.004a
no 10757 50
well 27 107
moderate 29 1613
poor 5931 28
Figure 2. The Kaplan–Meier survival analysis of gastric
adenocarcinoma patients (n=115) in the training group with
a low density of CD15+ + TINs (n=57) and a high density of
CD15+ + TINs (n=58). Based on the median number of CD15+ TINs
(21.60 cells/HPF) in the 115 cases, the patients were divided into two
groups: high CD15+ TINs group (§21.60 cells/HPF) and low CD15+ TINs
group CD15+ TINs (,21.60 cells/HPF). The survival rate of patients with
a high density of CD15+ infiltrating neutrophils was significantly lower
than that of patients with a low density of CD15+ infiltrating
neutrophils (Log rank test, p=0.002).
Intratumoral Neutrophils in Gastric Adenocarcinoma
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intratumoral neutrophils were a poor prognostic factor for
hepatocellular carcinoma following resection . Furthermore,
elevated neutrophil and monocyte counts in peripheral blood are
associated with poor survival in patients with metastatic melanoma
[23–24], small lung cell carcinoma , as well as gastric cancer
[26–28]. The results from these studies support our current
observations. In various carcinomas, neutrophils in the blood and
located in the tumor are poor prognostic features. These results
indicate that neutrophils play a negative role in various cancers
and may be a target for intervenient therapy.
In conclusion, we evaluated the prognostic significance of the
CD15+ TINs in a large number of clinical tissue specimens of
gastric adenocarcinoma by immunohistochemical analysis in the
current study. High level of CD15+ TINs in gastric adenocarci-
noma patients correlated to disease progression and poor clinical
outcome. CD15+ TINs was identified as an independent
prognostic factor for overall survival in patients with gastric
adenocarcinoma. Our findings suggest that CD15+ TINs is a
novel prognostic marker for gastric adenocarcinoma.
Materials and Methods
Patients and tissue specimens
Formalin-fixed, paraffin-embedded tissues were obtained from
gastric adenocarcinoma patients who underwent surgical resection
at the Sun Yat-sen University Cancer Center between 2002 and
2005. In the gastric adenocarcinoma patients, 115 cases were
assigned as the training group and 97 cases were assigned as the
test group. These patients did not receive preoperative chemo-
therapy or radiotherapy. Patients with autoimmune diseases were
excluded. Each tumor sample was assigned a histological grade
based on the World Health Organization (WHO) classification
criteria. All patients were staged using the 7th edition of the
International Union Against Cancer (UICC) Tumor-Node-
Metastasis (TNM) staging system. The follow-up dates of the
patients in this study are available and complete. Postoperative
follow-up occurred at our outpatient department and included
clinical and laboratory examinations every 3 months for the first 2
years, every 6 months during the third to fifth years, annually for
an additional 5 years or until patient death, whichever occurred
first. Overall survival, which was defined as the time from the
operation to the patient’s death or the last follow-up, was used as a
measure of prognosis. The characteristics of the gastric adenocar-
cinoma patients are shown in Table 3. All samples were coded
anonymously in accordance with local ethical guidelines (as
stipulated by the Declaration of Helsinki). The study was approved
by the Ethics Committee of Sun Yat-sen University Cancer
Center, and written informed consent was obtained from each
The paraffinic-embedded tissue blocks were sectioned at a
thickness of 2 mm for immunohistochemistry. The sections were
deparaffinized and rehydrated using graded ethanol. For the
antigen retrieval, the slides were immersed in EDTA (1 mmol/L,
pH 8.0) and boiled for 15 minutes in a microwave oven. After
Table 2. Univariate and multivariate analysis of overall survival in gastric adenocarcinoma patients in the training group.
Univariate analysisMultivariate analysis
HR 95% CI
p valueHR95% CIp value
Age (,60 v§60)
Gender (malevfemale)0.687 0.522–1.5250.676
Tumor size (,4 cmv§4 cm)2.392 1.170–4.8900.017a
Depth of invasion(T1-T2vT3-T4)6.4021.559–26.289 0.010a
Lymph node metastasis(N0vN1-N4)4.7562.147–10.538
Distance metastasis(novyes) 4.032 0.791–2.551
Histologic grade(well/moderate/poor) 1.0790.782–1.489 0.644
HR Hazard ratio, CI confidence interval.
Figure 3. The Kaplan–Meier survival analysis of gastric
adenocarcinoma patients (n=97) in the test group with a low
density of CD15+ + TINs (n=52) and a high density of CD15+ +
TINs (n=45). The patients were divided into high CD15+ TINs group
(§21.60 cells/HPF) and low CD15+ TINs group (,21.60 cells/HPF)
according to the median density of CD15+ TINs (21.60 cells/HPF), which
was obtained from the training group. Log-rank test shows that the
patients with the high density of CD15+ TINs showed significantly
poorer prognosis than those with the low density of CD15+ TINs
Intratumoral Neutrophils in Gastric Adenocarcinoma
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rinsing with PBS, endogenous peroxidase was blocked with 3%
hydrogen peroxide for 15 minutes at room temperature. The
slides were incubated with the primary mouse anti-CD15
monoclonal antibody (Zhongshan Golden Bridge Biotech, Beijing,
China) using a 1:100 dilution in a moist chamber at 4uC overnight.
After the primary antibody incubation, the slides were washed
with PBS three times. The sections were incubated with
horseradish peroxidase-conjugated secondary antibody (Zhong-
shan Golden Bridge Biotech, Beijing, China) for 30 minutes at
room temperature. Following this incubation, the slides were
washed three times in PBS. Finally, 3, 39-diaminobenzidine
tetrahydrochloride (DAB) was used to visualize the signal
development, and then, the sections were counterstained with
20% hematoxylin. The analysis was performed by two indepen-
dent observers using a Leica DM IRB inverted research
microscope (Leica Microsystems, Wetzlar, Germany) . The
tumor sections were screened at low power magnification (1006),
and the ten most representative fields were selected . In order
to evaluate the density of CD15+ TINs, each respective area of the
tumor nest was manually measured at 4006 high power
magnification [10,12,29]. The density of stained cells was
determined by computing the mean number of positively stained
cells per high powermicroscopic field (HPF) [10,12,29]. Based on
the median number of CD15+ TINs in the training group, the
patients were separated into high and low TINs groups. Each
sample was incubated using the same isotype antibody dilution
under the same experimental conditions as the negative control.
Quantitative values were expressed as the mean 6 SD or
median (range). The median value was used as a cut-off for the
subgroups of all immunohistochemical variables in our data. Chi-
squared tests were used to assess the relationship between CD15+
TINs and the clinicopathological features. Overall survival (OS)
was defined as the period from the initial diagnosis to death by any
cause. Overall survival curves were calculated by the Kaplan-
Meier method and analyzed by the long-rank test. Prognostic
factors were examined by univariate and multivariate analyses
using a Cox proportional hazards model. A two-sided p-
value,0.05 was considered statistically significant. All statistical
analyses were performed using SPSS software (version 16.0; SPSS
Inc., Chicago, IL, USA).
Conceived and designed the experiments: KP XDL JCX. Performed the
experiments: JJZ KP WW JGC. Analyzed the data: JJZ YHW LL JJL YBC
DDW QZP. Contributed reagents/materials/analysis tools: WW. Wrote
the paper: JJZ KP.
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Table 3. Clinical characteristics of gastric adenocarcinoma
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(%) Test Group (%)p value
,60 59 (51.3)61 (62.9)
§6056 (48.7) 36 (37.1)
Male 74 (64.3) 58 (59.8)
Female 41 (35.7)39 (40.2)
,4 32 (27.8) 25 (25.8)
§4 83 (72.2)72 (74.2)
Depth of invasion0.675
T1 5 (4.4) 3 (3.1)
T2 12 (10.4) 6 (6.2)
T3 25 (21.7)23 (23.7)
T473 (63.5)65 (67.0)
N037 (32.2) 25 (25.8)
N1 20 (17.4)21 (21.6)
N2 24 (20.9)21 (21.6)
N3 34 (29.5)30 (31.0)
Distance metastasis 0.075
M0 107 (93.0)83 (85.6)
M18 (7.0)14 (14.4)
UICC staging 0.366
I 13 (11.3)7 (7.2)
II30 (26.1)23 (23.7)
III63 (54.8) 53 (54.7)
IV9 (7.8) 14 (14.4)
Histologic grade 0.046
Well 27 (23.5) 14 (14.4)
Moderate 29 (25.2)17 (17.5)
Poor 59 (51.3) 66 (68.1)
No59 (51.3) 46 (47.4)
Yes 56 (48.7)51 (52.6)
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