Plasma cytokine levels and the presence of colorectal cancer
Plasma cytokine levels and the presence of colorectal cancer
Masaki YamaguchiID 0 3 4
Shin Okamura 0 3 4
Taiki Yamaji 3 4
Motoki Iwasaki 3 4
Shoichiro Tsugane 3 4
Vivek Shetty 2 3 4
Tomonobu Koizumi 3 4
0 Shinshu University, Graduate School of Science & Technology, Department of Mechanical Engineering & Robotics , Ueda, Nagano , Japan , 2 Division of Epidemiology, Center for Public Health Sciences, National Cancer Center, Tokyo, Japan, 3 Center for Public Health Sciences, National Cancer Center , Tokyo , Japan
1 ; 0.832 for the combination of IL-4 , IL-8, Eotaxin, IP-10 , USA
2 Section of Oral & Maxillofacial Surgery, UCLA Health Sciences Center , Los Angeles, CA , United States of America, 5 Shinshu University School of Medicine, Department of Comprehensive Cancer Therapy , Matsumoto, Nagano , Japan
3 9 , Eotaxin, G-CSF, and TNF-
4 Editor: Paul Proost, Katholieke Universiteit Leuven Rega Institute for Medical Research , BELGIUM
The levels of several plasma cytokines varied significantly between CRC patients and
control subjects, suggesting the possibility of differentially expressed plasma cytokines as
potential biomarkers for detecting the presence of CRC. Our results should be validated in
colorectal cancer (CRC).
. The Institutional
Review Board of the National Cancer Center will
evaluate the importance of their research proposal
considering the study participants? privacy by
following its prescribed process, and may provide
especial permission of access to a minimal dataset.
Funding: This work was supported in part by a
Fukushima grant for development of medical and
welfare devices, and development of low-cost,
rapid, and noninvasive diagnostic technology for
cancer using salivary biomarkers, Japan; and grant
no. 16H03166 from the Japan Society for the
Promotion of Science, for visualization of cancer by
cytokine coding methods and biosensor-arrays
with micro-capsules (P.I. M. Yamaguchi).
Colorectal cancer (CRC) is the second most common cancer occurring in females and the
third most common cancer in males worldwide [
]. The disease affects 1.36 million people
globally, accounting for nearly 10% of cancers [
]. CRC incidence and mortality continue to
increase, owing mainly to population aging, and possibly to factors including a ?westernized?
diet, lifestyle, and lack of health-care infrastructure and resources.
Numerous studies have been carried out to optimize the early detection, diagnosis and
treatment of this disease; CRC is one of the most-studied and best-characterized processes of
]. Current CRC screening methods can be generally grouped into invasive and
non-invasive tests. The non-invasive stool-based tests currently available include the
guaiacbased fecal occult blood test (gFOBT), the fecal immunochemical test (FIT), and the newer fecal
DNA test [
]. These tests are based on the concept of detecting blood or shredded cell debris
from vascularized polyps, adenomas and cancers. Owing to their simplicity and
user-friendliness, gFOBT and FIT have found widespread use in CRC screening but are burdened by low
sensitivity and specificity [
]. Invasive tests include flexible sigmoidoscopy and colonoscopy,
have higher sensitivity and specificity because they offer direct visualization and pathology
specimen collection [
]. However, routine use of such invasive tests for population-level screenings
is not very practical given the increased risk of complications, higher associated costs, and
limited capacity of the health-care system to perform such procedures. Furthermore, the logistics
and discomfort involved with colonoscopies can cause at-risk patients to choose forgo screening
entirely. Clearly, there is a need for alternates to gFOBT and FIT with higher sensitivity and
specificity and are relatively inexpensive and straightforward to perform.
The ability to screen blood samples for tumor-related biochemicals has generated
considerable interest in their use for detecting the presence of CRC early in its development. The hope
is that the convenience of screening blood tests would allow for early diagnosis and treatment
and lead to significant reductions in cancer-related morbidity and mortality [
]. Of the various
pathways to tumor formation and progression, the inflammatory pathway has stimulated
particular attention [
]. Several excellent reviews have described the cellular and molecular roles
of inflammation in the development of cancer [
]. A central feature of activated immune
cells is the production and release of growth factors and cytokines that modulate the
inflammatory milieu in tumor tissues. Currently, more than 300 different cytokines have been
identified. CRC has been linked to systemic and local changes in the cytokine profile [
recent work indicates that multiple pro-tumorigenic and also antitumorigenic cytokines are
differently expressed in distinct CRC tissues [
]. Chemokines, small peptides that are
structurally and functionally similar to growth factors, are also among the key players that promote
cancer cell metastasis in some types of cancers. Chemokine ligand-receptor interactions have
been reported to be involved in CRC progression [
]. Thus, the differential expression of the
blood cytokines could have the potential in the early detection of CRC. To explore this, we
carried out a multicenter, hospital-based case-control study to examine the associations between
plasma cytokine levels and the presence of CRC. We hypothesized that the levels of some
plasma cytokines would fluctuate depending on the presence of CRC and combinatorial
cytokines would have greater discriminant ability.
Material and methods
Details of the Nagano CRC study have been described elsewhere [
]. In brief, a multicenter,
hospital-based case-control study was conducted at four hospitals in Nagano Prefecture, Japan
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(Matsushiro General Hospital, Saku General Hospital, Shinonoi General Hospital, and
Hokushin General Hospital) between 1998 and 2002. Eligible cases were patients newly
diagnosed with CRC during the study period at those hospitals. For each subject identified with
CRC, the healthy controls (verified to be CRC-free) matched for age (? 3 years) and gender
were randomly selected from the hospital?s health checkup program. All study subjects gave
written informed consent for their participation in the study. Subjects completed a
self-administered questionnaire concerning general and lifestyle characteristics (e.g. age, gender, height,
weight, smoking, and drinking), as well as personal and family medical history, and provided
blood samples before their treatment (CRC cases) or during their health checkup (healthy
controls). After excluding nine ineligible cases (one mucinous carcinoma case, one squamous cell
carcinoma case, and seven cases without an available blood sample) and their matched
controls, 113 cases and 226 controls were included in the Nagano CRC study.
For the present study of plasma cytokines, we selected all of the 66 CRC cases (36 males and
30 females) aged 50?69 years with no current smoking and all of the 87 healthy controls (51
males and 36 females) within the same age range as cases and without current smoking,
cancer, or surgery. Our study was approved by the Ethics Committee of Shinshu University School
of Medicine (No.3361) and National Cancer Center (T2011-005) of Japan.
Multi-analysis of cytokines
Collected blood samples were stored at ?80?C and just prior to analysis thawed at 4?C in a
refrigerator. Finally, the blood samples were brought to room temperature (24?C) before being
added to the assay plate. After that, the blood samples were centrifuged at 1,500 ? g for 15 min.
A micropipette was then used to sample a fixed aliquot of each sample (50 ?L) for subsequent
We used a multiplex bead array assay (Bio-Plex) that has previously been used to examine
plasma cytokines [
]. Twenty seven cytokines (interleukin (IL)-1ra, IL-1?, IL-2, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-10, IL12p70, IL-13, IL-15, IL-17A, C-C motif chemokine ligand 11
(CCL11; Eotaxin), fibroblast growth factor 2 (FGF-2), colony stimulating factor 3 (CSF3;
G-CSF), colony stimulating factor 2 (CSF2; GM-CSF), interferon gamma (IFN-?), tumor
necrosis factor alpha (TNF-?), C-X-C motif chemokine ligand 10 (CXCL10; IP-10), C-C motif
chemokine ligand 2 (CCL2; MCP-1), C-C motif chemokine ligand 3 (CCL3; MIP-1?), C-C
motif chemokine ligand 4 (CCL4; MIP-1?), platelet-derived growth factor-BB (PDGF-BB),
regulated on activation, normal T cell expressed and secreted (RANTES), and vascular
endothelial growth factor (VEGF)) were analyzed according to the manufacturer?s instructions. The
Bio-Plex multiplex bead array immunoassay system used human cytokine panels and the
plates were read on a Bio-Plex Array Reader (Bio-Plex 200 System and Bio-Plex Manager
Version 6.1, Bio-Rad Laboratories, Inc., Tokyo, Japan).
The results from the samples and their clinical information were entered into a structured
database by research staff not directly involved in patient diagnosis, treatment, or sample
examination. Patient diagnostic and pathological data were de-identified during sample
collection and biomarker detection.
All the statistical analyzes were performed with the Statistical Package for Social Sciences
(SPSS) version 25 (Advanced Analytics, Inc. Tokyo, Japan). Unless otherwise stated,
continuous data are summarized as the mean ? standard deviation (SD). A value of p < 0.05 was
taken to represent statistical significance. The statistical analysis was not performed if the
number of data points missing from one group exceeded half of all data points. In the statistical
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analysis, the plasma cytokine levels were subjected to log-transformation as well as
standardizing, those less than the limit of detection were given a value of half the limit of detection
Prior to the construction of a prediction model, we compared mean plasma levels of each
cytokine between cases and controls by using a multiple linear regression analysis with
adjustment for gender, age, and hospital.
Next, a multiple logistic regression analysis was conducted to examine the association
between plasma cytokines and the presence of CRC after adjustment for gender, age, and
hospital. Odds ratios (OR) and their 95% confidence intervals (95% CI) for the presence of CRC
were estimated for each of the plasma cytokines.
A correlation analysis was also performed for all of the pairwise of cytokines to evaluate
For the construction of a prediction model, candidate plasma cytokines were selected
according to the following procedures. First, we nominated as the initial candidate the plasma
cytokine that showed the smallest p-value in the aforementioned multiple logistic regression
analysis, and excluded all cytokines whose correlation coefficients with the selected cytokine
exceeded 0.7. Then, we identified another candidate cytokine with the next smallest p-value,
and eliminated some of the remaining cytokines in the case where their correlation coefficients
with the second selected cytokine were above 0.7. These procedures were repeated until
candidate cytokines were finalized. By applying a backward elimination method to a logistic
regression model with all possible candidates of plasma cytokines and fixed variables of gender, age,
and hospital, we constructed a prediction model for the presence of CRC .
The model performance was assessed by a discrimination test using receiver operating
characteristic (ROC) analysis . ROC curves were depicted to investigate the discriminatory
power of the plasma cytokine levels. The areas under the curves (AUC) were calculated to
provide an overall summary of the detection accuracy of the plasma cytokine levels, and were
empirically classified into three levels: poor when 0.50 AUC < 0.69, good when
0.70 AUC < 0.89, and excellent when 0.90 AUC < 1.
Multi-analysis of plasma cytokines in the CRC patients and controls
all 13 plasma cytokines, except IFN-? and RANTES, showed an increasing trend according to
tumour progression (S1 Table and S1 Fig). The associations of the above 13 plasma cytokines
with the presence of CRC were all statistically significant (Table 3).
Construction of a prediction model for the presence of CRC
As seen in Table 3, IL-9 and IL-4 showed the smallest p-values (< 0.00001) and were selected
as the initial candidates for the IL-9 and IL-4 models, respectively. Seven plasma cytokines
were nominated as additional candidates for each model: Eotaxin, G-CSF, TNF-?, IP-10,
MIP-1?, MIP-1?, and PDGF-BB for the IL-9 model; and IL-8, Eotaxin, TNF-?, IP-10,
MIP1?, MIP-1?, and PDGF-BB for the IL-4 model. Finally, the backward elimination method
revealed two multivariable prediction models (logistic models) for the presence of CRC as
follows (p < 0.05, Table 4):
IL 9 model : z
IL 4 model : z
? 1:643 IL 9 ? 0:743 Eotaxin ? 0:842 G
0:014 age ? 0:989 hospital B
? 0:145 hospital C ? 0:071 hospital D
1:416 TNF a
1 The multiple linear regression analysis was conducted after adjustment for gender, age, and hospital.
2The total number of plasma cytokine levels which were less than the limit of detection (LOD). The LOD was 0.02 pg/mL for IL-6, 0.13 pg/mL for IL-10, 0.18 pg/mL for
IL-17A, 0.89 pg/mL for FGF-2, and 0.10 pg/mL for VEGF.
3Significant at p < 0.05.
ROC analysis of a prediction model for the presence of CRC
We performed ROC analysis to closely evaluate the above two prediction models for the
presence of CRC (Fig 2). In the IL-9 model, the AUC, sensitivity, and specificity of the optimal
cutoff points were 0.819, 0.652, and 0.839, respectively, while the corresponding values in the IL-4
model were 0.832, 0.742, and 0.767, respectively. Both models thus demonstrate ?good?
capability for discriminating between CRC patients and controls. A comparative prediction model
without cytokines (model 0) was constructed by using gender, age, and hospital as variables.
Its AUC, sensitivity, and specificity of the optimal cut-off points were 0.559, 0.485, and 0.655,
respectively, therefore, the model demonstrated ?poor? capability. All of the evaluation
parameters for the IL-9 and IL-4 models were better than those for model 0. Also, single-cytokine
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Fig 1. Comparison of plasma cytokine levels between CRC patients and controls, significant differences were
observed by multiple linear regression analysis.
models presented lower AUCs (0.657?0.755) than the multi-cytokine IL-9 and IL-4 models
The study results supported our working hypothesis that the levels of some plasma cytokines
vary depending on the presence of CRC. Even after controlling for gender, age, and hospital,
the plasma levels of CRC patients and controls differed significantly in terms of the following
13 cytokines: IL-4, IL-8, IL-9, IL-17A, Eotaxin, G-CSF, IFN-?, TNF-?, IP-10, MIP-1?, MIP-1?,
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95% confidence interval
1.895 - 4.967
1.163 - 2.476
2.002 - 5.364
1.151 - 2.914
1.320 - 3.039
1.468 - 3.417
1.022 - 2.099
1.077 - 2.250
1.233 - 2.532
1.144 - 2.404
1.215 - 2.507
1.460 - 3.607
1.028 - 2.288
PDGF-BB, and RANTES (Table 2 and Fig 1, p < 0.05). The combinatorial assessment of some
of these plasma cytokines showed promise for detecting the presence of CRC. In fact, the ROC
analysis showed that the IL-9 and IL-4 models had ?good? capability for discriminating
between CRC patients and controls (Table 4 and Fig 2). These two models showed similar
AUC values, although some differences were observed; the performance of the IL-9 model was
excellent with respect to specificity, while the IL-4 model balanced both sensitivity and
specificity (Table 5).
In our research, the logistic regression analysis revealed that the cytokines IL-9 and IL-4
had potential association with the presence of CRC (Table 3). IL-9 is a cytokine produced by
CD4+ Th2 cells as well as by some B lymphomas; it has been shown to induce an increase in
the proliferation of CRC cells and to promote tumorigenesis in CRC cells . Kantola et al.
conducted a screening cohort study using cytokines in serum, including IL-9, and concluded
that cytokine biomarkers might be a promising tool for the detection of CRC . Broadly,
IL4 can be categorized as a type of anti-inflammatory cytokine. IL-4 and IFN-? are the most
frequently described cytokines in the inflammatory process. Szylberg et al. analyzed 144
colorectal polyps and showed a significantly increased level of IL-4 in adenomas, serrated adenomas,
and hyperplastic polyps compared with the control group . Sharp et al. reported that levels
Fig 2. ROC analysis of the presence of CRC using a panel of select plasma cytokines that were most associated
with CRC. (A) IL-9 model and (B) IL-4 model.
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Area Under the ROC Curve
of the anti-inflammatory cytokine IL-4 were significantly elevated in advanced CRC, whereas
IFN-? levels were not statistically different . Recent studies have found that IL-4 levels in
colorectal polyp-derived serum were significantly higher than those in serum from healthy
Other cytokines included in the IL-9 and IL-4 models warrant mention. There is evidence
that the cytokine Eotaxin is strongly associated with primary and metastatic tumors of
colorectal origin . Both IL-4 and IL-13 synergistically enhance TNF-??induced Eotaxin
production . Natori et al. suggested that G-CSF may have the potential to promote tumor growth,
at least in part, by stimulating angiogenesis . The level of G-CSF has been examined in
CRC patients and found to be significantly higher than in healthy subjects . Despite the
small sample size, another study found significantly higher levels of G-CSF in CRC patients
before surgery compared with controls at baseline . TNF-? is a potent pro-inflammatory
cytokine thought to be involved in the pathogenesis of inflammatory bowel disease  and
has been reported to promote inflammation and colitis-associated cancer . In the blood of
the patients with CRC, a significant elevation has been reported in the levels of TNF-? [36,37].
Dimberg et al. analyzed 50 CRC patients and found a significantly higher IL-8 (CXCL 8) level
in cancer tissue compared with paired normal tissue, and showed that CRC patients exhibited
significantly higher plasma levels than healthy controls . Crucitti et al. conducted screening
of 30 CRC patients; although the sample size was small, significantly higher levels of IL-1?,
IL7, IL-8, G-CSF, IFN-?, and TNF-? were detected in CRC patients compared with controls at
baseline . The CXCL10 (IP-10)/CXCR3 axis of inflammatory mediators is one of the most
important chemokine axes and has been proven to be a lymphocyte-associated metastasis
mediator in several tumors , although in one, report serum levels of IP-10 were of
measureable concentration but did not differ between the control and CRC groups .
Several studies have investigated validity of the Bio-Plex multiplex bead array immunoassay
system (originally developed by Luminex Co.) by comparison with traditional enzyme-linked
immunosorbent assay (ELISA) [
]. Carson et al. conducted a simultaneous quantitation
of 15 cytokines using the multiplex immunoassay system in parallel with quantitation by
ELISAs, and showed that use of the multiplex bead array did not reduce sensitivity for any
cytokines including IL-1?, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, GM-CSF, IFN-?, TNF-?, and
MCP-1 compared with that of the ELISAs . Prabhakar et al. indicated that overall values of
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the cytokines IL-1?, IL-6, IL-8, and TNF-? were not significantly different between the two
types of assays [
]. We performed a similar validation study by measuring IL-9, and
calculated a Spearman?s correlation coefficient of 0.47 between the Bio-Plex multiplex bead array
and ELISA (unpublished data). Compared with ELISA, the Bio-Plex system showed a more
appropriate range of circulating IL-9 concentrations, which is in line with the previous reports
]. Although the multiplex bead array immunoassay has emerged as a suitable option for
measuring several plasma cytokines at once, extrapolation of our results should be made with
caution, particularly with regard to plasma cytokine data obtained by other assays.
In our study, the levels of several plasma cytokines varied significantly between CRC
patients and control subjects, suggesting the possibility of the differential expression of plasma
cytokines as potential biomarkers for detecting the presence of CRC. Our results should be
validated in other populations and should be supported by future studies to examine the temporal
changes in plasma cytokine levels according to colorectal cancer progression and prognosis.
Lengthy and careful consideration should be given why IL-9 and IL-4 strongly correlated with
the presence of CRC.
S1 Table. Results of multi-analysis of cytokines; comparisons between stage 0 ?II patients
(42 cases; 23 males and 19 females), stage III?IV patients (24 cases; 13 males and 11
females), and controls by multiple linear regression analysis. Significant differences were
observed in mean plasma levels for IL-4, IL-8, IL-9, IL-17A, Eotaxin, G-CSF, TNF-?, IP-10,
MIP-1?, MIP-1?, and PDGF-BB between CRC patients and controls (p < 0.05).
S1 Fig. Comparison of plasma cytokine levels between stage 0 ?II patients (42 cases; 23
males and 19 females), stage III?IV patients (24 cases; 13 males and 11 females), and
controls. Significant differences were observed for IL-4, IL-8, IL-9, IL-17A, Eotaxin, G-CSF,
TNF?, IP-10, MIP-1?, MIP-1?, and PDGF-BB (p < 0.05) using multiple linear regression analysis.
We thank the patients and healthy volunteers for their support and participation in this study.
Conceptualization: Masaki Yamaguchi.
Data curation: Masaki Yamaguchi, Shin Okamura, Taiki Yamaji, Motoki Iwasaki.
Funding acquisition: Masaki Yamaguchi.
Investigation: Masaki Yamaguchi, Shin Okamura, Taiki Yamaji, Motoki Iwasaki.
Resources: Taiki Yamaji, Motoki Iwasaki, Shoichiro Tsugane.
Supervision: Shoichiro Tsugane, Vivek Shetty, Tomonobu Koizumi.
Writing ? original draft: Masaki Yamaguchi.
Writing ? review & editing: Taiki Yamaji, Motoki Iwasaki, Shoichiro Tsugane, Vivek Shetty,
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