An increased number of negative lymph nodes is associated with a higher immune response and longer survival in colon cancer patients
Cancer Management and Research
An increased number of negative lymph nodes is associated with a higher immune response and longer survival in colon cancer patients
Wen-Zhuo He 2 3
Wan-Ming Hu 1 2
Peng-fei Kong 2 3
Lin Yang 2 3
Yuan-Zhong Yang 1 2
Jiang 2 3
Chen-Xi Yin 2 3
Juan Qiu 2 3
Zhang 1 2
Bei Zhang 2 3
Ping Xia 2 3
0 Department of Pathology, School of Basic Medical Sciences, Southern Medical University , Guangzhou , People's Republic of China
1 Department of Pathology, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine , Guangzhou, Guangdong , People's Republic of China
2 who underwent surgery at Sun Yat-sen University Cancer Center between 2009 and 2014 were
3 VIP Region, Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine , Guangzhou, Guangdong , People's Republic of China
4 Department of Pathology, Nanfang Hospital, Southern Medical University , Guangzhou , People's Republic of China
8 1 0 2 - l u J - 2 1 n o 9 1 1 . 9 5 . 2 3 . 3 1 2 y b / m o c . s s e r p e .vdo l.yn w o /ww seu :/s la tp n th rso froedm rpeoF PowerdbyTCPDF(ww.tcpdf.org) *These authors contributed equally to this work Background: The purpose of the present study was to examine the relationship among the number of negative lymph nodes (LNs), the local and systemic immune response, and survival in patients with colon cancer. Patients and methods: One thousand one hundred and fifty-seven patients with colon cancer included. We examined negative LNs in relation to the local and systemic immune response, including percentage carcinoma, neutrophil and lymphocyte infiltration, Crohn's-like reaction, neutrophil to lymphocyte ratio, platelets, and C-reactive protein (CRP). Disease-free survival and overall survival were also examined. We performed subgroup analysis based on the distribution of negative LNs. Results: An increased number of negative LNs was associated with greater neutrophil invasion (p=0.001), more lymphocyte invasion (p=0.001), and more Crohn's-like reaction (p=0.001). No significant correlation was observed between negative LNs and the neutrophil to lymphocyte ratio. More than 12 negative LNs were associated with increased platelets and CRP levels. A higher number of negative LNs was independently associated with longer disease-free survival in stage I+II patients (p=0.004) and stage III patients (p=0.015), while negative LNs were also independent prognostic factors in stage IV patients (p=0.007). Conclusion: Our study suggests that negative LNs are indicators of the immune response and are associated with a better prognosis in patients with colon cancer.
colon cancer; negative lymph nodes; immune response; survival
open access to scientific and medical research
Lymph node (LN) metastasis has important prognostic implications in patients with
cancer.1 Recently, the number of negative LNs has also attracted attention for its
prognostic value. It has been reported that an increased number of negative LNs was
associated with longer survival in breast cancer,2,3 lung cancer,4 gastric cancer,5,6
esophageal cancer,7 cervical cancer,8 and colorectal cancer.9–12 The mechanism underlying
the relationship between the number of negative LNs and the patient survival remains
elusive. One possible reason is that increased numbers of negative LNs indicate
accurate staging and higher care quality.9 However, Zhuo et al reported that negative LNs
are also prognostic factors in metastatic gastric cancer,5 which cannot be explained
by accurate staging or surgical intervention.
Another possible explanation for the prognostic value of negative LNs is that the
increased negative LN number indicates higher tumor and host immune interaction.
Until now, little evidence has been reported to support this hypothesis.12 Therefore,
we performed this study to evaluate the relationship between
negative LNs and the host immune response, including
both the local and the systemic tumor environment. We also
studied the prognostic value of negative LNs in patients with
colon cancer. Moreover, we performed subgroup analysis
based on tumor stage in order to minimize its impact on our
assessment of patients’ survival.
-Ju2 Patients and methods
on Patient selection
.119 The data set of Sun Yat-sen University Cancer Center was
.952 built prospectively. The records were retrospectively
ana.313 lyzed. Patients who met the following criteria were enrolled:
y2 1) diagnosed with colon cancer between 2009 and 2014;
/m 2) underwent primary tumor resection; 3) available records
.cso of routine blood tests before any treatment; 4) available
rpe paraffin-embedded tissue blocks; and 5) available follow-up
.vdow l.yno itinofno,rmhyaptieornp.yTrehxeieax,chleumsiaotnolcorgitiecraial dwiesreeaspea,tieennttesrwobitrhosiinsf,eoc-r
Measurement of neutrophil,
lymphocytes, platelets (PLT), CRP, and
carcinoembryonic antigen (CEA)
Neutrophil, lymphocytes, and PLT were measured using
routine blood tests. These tests were conducted using the
Sysmex XE-5000™ Automated Hematology System (Shanghai,
People’s Republic of China). CRP was measured using the
Hitachi Ltd. Automatic Analyzer 7600-020 (Tokyo, Japan).
CEA was evaluated using electrochemiluminescence with the
Hoffman-La Roche Ltd. Elecsys 2010 Chemistry Analyzer
(Basel, Switzerland). The neutrophil to lymphocyte ratio
(NLR) was categorized into two groups (>3 and ≤3).
Assessment of mismatch repair (MMR)
Immunohistochemistry for the four most common MMR
proteins was performed with the standard Envision (Dako
Denmark A/S, Glostrup, Denmark) two-step procedure.
The slides were dried overnight at 37°C, dewaxed in xylene,
rehydrated through graded alcohol, and immersed in 3%
hydrogen peroxide for 20 minutes to block endogenous
peroxidase activities. They were then pretreated in antigen
retrieval buffer (EDTA buffer, pH 8.0, 100°C, 2 minutes in a
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pressure cooker), and incubated with 10% normal goat serum
at room temperature for 10 minutes to reduce nonspecific
binding. Subsequently, the slides were incubated overnight
at 4°C using the following antibodies: MLH1 (1:50; Beijing
Zhong Shan Golden Bridge Biological Technology, Beijing,
People’s Republic of China), PMS2 (1:50; Beijing Zhong
Shan Golden Bridge Biological Technology), MSH2 (1:50;
Beijing Zhong Shan Golden Bridge Biological Technology),
and MSH6 (1:50; Beijing Zhong Shan Golden Bridge
Biological Technology). After rinsing five times with 0.01 mol/L PBS
(pH=7.4) for 10 minutes, detection of the primary antibody
was performed using a secondary antibody (Envision) for 1
hour at room temperature and stained with diaminobenzidine
(DAB) after washing in PBS again. Finally, the sections were
counterstained with Mayer’s hematoxylin, dehydrated and
mounted. PBS was used instead of the primary antibody
in the negative control. Nonneoplastic colonic mucosa,
stromal cells, and infiltrating lymphocytes or the centers of
lymphoid follicles were used as internal positive controls.
Known MMR-deficient (dMMR) colon carcinomas served
as external negative controls. Two experienced pathologists
evaluated the staining results independently and without
any prior knowledge of the patients’ clinical data. Normal
expression was defined as nuclear staining within tumor
cells, whereas negative protein expression was defined as
the complete absence of nuclear staining within tumor cells
with concurrent internal positive controls. Tumors with the
loss of MLH1/PMS2/MSH2/MSH6 proteins, as visualized
using light microscopy, were classified as MLH1/PMS2/
MSH2/MSH6 negative. If internal nonneoplastic tissues
showed invalid negative staining, the procedure was repeated.
When the opinions of the two pathologists were different, an
agreement was reached by careful discussion.
Assessment of local tumor
The carcinoma percentage, neutrophil infiltration (both in
the central region and at the invasive margin), lymphocyte
infiltration (both central region and invasive margin), and
Crohn’s-like reaction were assessed as previously reported13–15
by two pathologists who were blinded to the clinical data.
Inflammatory cell reactions (neutrophil and lymphocytes)
were estimated in H&E-stained sections by studying the
central areas of the tumor and at the invasive margin. Four to
six sections were routinely evaluated per tumor. We evaluated
all of the sections and those with the most invasive part of the
primary tumor were chosen to avoid bias. Dr Wan-Ming Hu
and Dr Hui-Zhong Zhang assessed these markers. When the
opinions of the two pathologists were different, agreement
was reached by careful discussion.
between negative LNs and the NLR. More than 12 negative
LNs were associated with increased PLT and CRP levels.
Patient follow-up and statistical analysis
Disease-free survival (DFS), defined as the time from
diagnosis to the time of the first event (loco-regional recurrence,
metastasis, or death), and overall survival (OS), defined as
the time from diagnosis to the date of death or the date of
last follow-up, were examined in this study. The date of last
follow-up was December 31, 2016.
All statistical analyses were performed using SPSS
version 22. Frequencies and descriptive statistics were used to
compare patient characteristics. Chi-square test was used to
detect difference. Survival curves were calculated with the
Kaplan–Meier method, and the differences were compared
using the log-rank test. Multivariate analysis using a Cox
proportional hazards model was used to test independent
signif icance by backward elimination of insignif icant
explanatory variables. A p-value of <0.05 was considered
Seven hundred and thirteen men and 444 women were
included. The median age was 59 (range from 19 to 87) years.
Four hundred and thirty-five (37.6%) patients had right-sided
colon cancer, and 703 (60.8%) had left-sided colon cancer.
One hundred and six patients (9.2%) had stage I disease,
504 (43.6%) had stage II, 264 (22.8%) had stage III, and 283
(24.4%) had stage IV. The mean total LN counts was 15.9.
The mean negative LN counts was 14.5. The distributions
of clinicopathological features and their relationship with
negative LNs are shown in Table 1. Increased negative LNs
were associated with right-sided colon cancer (p=0.001),
well or moderately differentiated disease (p=0.005), a lower
CEA level (p=0.001), dMMR status (p=0.005), less neural
invasion (p=0.001), and venous invasion (p<0.001).
Negative LNs, the local tumor
environment, and systemic inflammatory
The relationship between negative LNs, the local tumor
microenvironment, and the systemic inflammatory response
is shown in Table 2. An increased number of negative LNs
was associated with greater neutrophil invasion (p=0.001),
higher-grade lymphocyte invasion (p=0.001), and more
Crohn’s-like reactions (p=0.001). For the systemic
inflammatory response, no significant correlation was observed
Impact of negative LNs on recurrence
Since only 106 patients had stage I disease, we grouped them
together with the 504 patients with stage II disease. Among
the patients with stage I and stage II disease, 58 experienced
recurrence or distant metastasis during the follow-up time.
Seven of 35 (20.0%) patients with 0–3 negative LNs, 7/50
(14.0%) patients with 4–6 negative LNs, 20/167 (12.0%)
patients with 7–12 negative LNs, and 24/358 (6.7%) patients
with ≥13 negative LNs experienced recurrence or distant
metastasis. As shown in Figure 1, an increased number of
negative LNs was associated with longer DFS (p=0.036). In
multivariable analyses, the impact of negative LNs (p=0.004)
was independent of age, sex, primary tumor location, T stage,
differentiation, venous invasion, neural invasion, MMR
status, neutrophil infiltration, lymphocyte infiltration, CEA
level, CRP level, and the NLR (Table S1). Among patients
with stage III disease, 6/14 (42.9%) patients with 0–3
negative LNs, 17/36 (47.2%) patients with 4–6 negative LNs,
22/85 (25.9%) patients with 7–12 negative LNs, and 21/129
(16.3%) patients with ≥13 negative LNs experienced
recurrence or distant metastasis. As shown in Figure 1, an increased
number of negative LNs was a predictor of longer DFS
(p<0.001). In multivariable analyses, the impact of negative
LNs (p=0.015) was also independent of age, primary tumor
location, T stage, differentiation, venous invasion, neural
invasion, MMR status, neutrophil infiltration, lymphocyte
infiltration, CEA level, CRP level, and the NLR (Table S2).
For patients with stage IV disease, the median OS was
19.8 months for those with 0–3 negative LNs, 23.2 months
for those with 4–6 negative LNs, 23.2 months for those with
7–12 negative LNs, and 27.1 months for those with ≥13
negative LNs. Similarly, an increased number of negative
LNs was associated with longer OS (p<0.001). In
multivariable analyses, the impact of negative LNs (p=0.007) was an
independent factor for OS (Table S3).
Subgroup analysis by location of LN
Previous studies reported that the location of positive LNs
has an important prognostic value.16 However, the impact of
the distribution of negative LNs has not been fully studied.
Thus, we performed subgroup analysis by grouping LNs into
pericolic nodes, intermediate nodes, and nodes at the origin
of the inferior mesenteric artery. Associations were observed
between negative LNs and the inflammatory response, even
Notes: #One patient had missing CEA values. *Chi-square test was used to compare patient characteristics, and a p-value <0.05 was considered significant.
Abbreviations: CEA, carcinoembryonic antigen; MMR, mismatch repair; dMMR, MMR-deficient; pMMR, MMR-proficient.
when the distribution of negative LNs was taken into account
(Tables S4–S9). However, no association was observed
between age and the number of negative pericolic nodes
(p=0.351) nor was any association observed between the NLR
and the number of negative intermediate nodes (p=0.196).
We found that the number of negative pericolic nodes had no
prognostic value, whereas an increased number of negative
intermediate nodes were associated with better survival in
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Note: *Chi-square test was used to compare patient characteristics, and a p-value <0.05 was considered significant.
Abbreviations: NLR, neutrophil to lymphocyte ratio; PLT, platelets.
patients with all stages of colon cancer (Figure S1). Increased
numbers of negative nodes at the origin of the inferior
mesenteric artery were only associated with longer survival in
patients with stage IV colon cancer (p=0.006, Figure S1).
Negative LNs have been reported as prognostic factors in
colon cancer, gastric cancer, breast cancer, esophageal
cancer, and cervical cancer.2–4,6–9,11,12 In this study, we confirmed
a significant correlation between the number of negative
LNs and the survival of patients with colon cancer, even in
metastatic colon cancer. The correlation was independent of
tumor stage and other routine clinicopathological features.
More importantly, we have provided clear evidence that an
increased number of negative LNs was also an indicator of
greater neutrophil and lymphocyte infiltration, both in the
invasive margin and in the central region of tumor
microenvironment. Negative LNs were also a predictor of a Crohn’s-like
reaction in patients with colon cancer.
The mechanism underlying the relationship between the
number of negative LNs and the survival remains unclear.
Several hypotheses have been proposed. One possible reason
is that assessing the numbers of negative LNs helps to reduce
the likelihood of misclassification of stage III disease as stage
II.9 Furthermore, increased numbers of negative LNs might
be an indicator of better therapy, including both surgical
treatment and pathological assessment. However, the survival
advantage associated with negative LNs in patients with
metastatic colon cancer cannot be explained by these reasons.
An alternative explanation is that an increased number
of negative LNs indicates a stronger immune reaction to the
tumor, which is a well-known predictor of a better
prognosis.12 Once the immune system detects a tumor, the local LNs
become enlarged in response. Enlarged LNs can be more
easily detected surgically and pathologically. However, little
evidence to date has supported this hypothesis. By analyzing
the tumor microenvironment, we found that negative LNs are
correlated with local neutrophil and lymphocyte infiltration,
which supports this hypothesis. Furthermore, patients with
increased numbers of negative LNs are more likely to be
young, have colon cancer on the right side, have a larger
primary tumor, and have dMMR status. These
characteristics are associated with a higher lymphocytic reaction to the
tumor. A better understanding of this interaction would lead
to improved risk stratification and therapeutic intervention.
Recently, the concept of negative LNs has attracted
increased attention in various types of cancer.17–19 Ahmadi
et al observed that LN yield and negative LNs were influenced
by patient age, site of disease, and T stage in patients with
colorectal cancer.20 By analyzing 1,167 patients with
colorectal cancer, Tsai et al found that age, tumor size, and higher
T stage were independent factors affecting the examinations
of LN.21 Zhang et al reported a higher predicted accuracy of
survival through the incorporation of negative LN into
American Joint Committee on Cancer stages.22 Others presumed the
immune response and the node count are interrelated, since
antitumor immune response may lead to the enlargement of
LNs and facilitate detection.23 Those studies, together with
our finding, highlight the importance of negative LNs in
China (81272641 and 81572409). We have engaged the
serpatients with colorectal cancer.
vices of Editage [www.editage.cn], an English language
editGuinney et al classified most colon cancers into four
ing service, to improve the language of the revised manuscript.
consensus molecular subtypes (CMSs).24,25 The CMS1 group,
including most dMMR colon cancers, is characterized by
right-sided lesions, a higher histopathological grade,
enrichThe authors report no conflicts of interest in this work.
ment of BRAF mutations and strong immune cell infiltration,
particularly T lymphocytes.24 Notably, patients with ≥13
negative LNs also have these characteristics. Ogino et al also
observed a positive correlation between BRAF mutations and
increased numbers of negative LNs.12 Although we did not
classify patients into different CMS types, it is possible that
patients with CMS1 colon cancer have more negative LNs,
indicating that immune classification is associated with tumor
Li et al reported that the negative to positive LN ratio
was a superior prognostic factor in patients with stage III
colorectal cancer.10 In this study, we did not evaluate the ratio
since it was also influenced by the number of positive LNs.
Our study suggests that LNs could provide more prognostic
information besides N stage and require more attention in
This study has several limitations. First, it is a
retrospective study in a single institute; thus, the results should be
interpreted with caution. Second, we determined the MMR
status using only immunohistochemistry, and we did not use
PCR. Data regarding several important molecular features,
including RAS and BRAF mutations, were also absent since
these factors are not routinely tested in our institute. Third,
only DFS was analyzed in patients with stages I, II, and III
disease since OS had not been reached yet. Fourth, due to the
retrospective nature of this study, we do not know the name
of the Dr who harvested the LNs. The size of mesentery was
also unavailable. Those factors could lead to bias in our data.
In conclusion, our study suggests that negative LNs are
indicators of immune response and are associated with a
better prognosis in patients with colon cancer.
Availability of data
The data of the study were deposited in the Research
Data Deposit system of Sun Yat-sen University Cancer
This work was supported by grants from the Natural Science
Foundation of Guangdong, China (2015A030313010),
Science and Technology Program of Guangzhou, China
(1563000305), and National Natural Science Foundation of
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