Keywords
gallbladder cancer - interleukin-6 - tumor necrosis factor-alpha
Introduction
Gallbladder cancer (GBC) is the most common lethal malignancy among biliary tract
cancers worldwide.[1] In India, GBC is among the top 20 cancers, with a prevalence of 1.82 per 100,000.[2] It is found to be more prevalent in female residents of the Indo-Gangetic belt in
Northern India.[3] Usually, GBC has a poor prognosis due to delayed diagnosis because of nonspecific
symptoms.[3] To date, there are no definitive tumor markers or screening tests for early diagnosis
or determining the prognosis of GBC.
GBC is associated with many ethnic and genetic factors.[4] Chronic inflammation due to gallstones is one of the major risk factors for GBC.[5] The link between inflammation and cancer is well documented. Interleukin-6 (IL-6)
and tumor necrosis factor-alpha (TNF-α) are both considered the primary mediators
of a network of interactive signals. IL-6 and TNF-α are proinflammatory cytokines
involved in the pathogenesis of various cancers, with pleiotropic activities including
inflammatory response as well as lymphangiogenesis, tumorigenesis, metastasis, cell
proliferation, differentiation, and apoptosis.[6]
IL-6 is secreted by monocytes, Kupffer's cells, keratinocytes, endothelial cells,
and B- and T-cells. IL-6 signaling is mediated through the Janus kinases (JAK)/signal
transducer and activator of transcription proteins (STAT) pathway and leads to the
expression of molecules involved in proliferation, angiogenesis, and metastasis.[6] Increased IL-6 expression has been observed in prostatic, lung, breast, and ovarian
cancer.[7]
[8]
[9]
[10] One study by Liu et al showed higher circulating levels of IL-6 in GBC, suggesting
further exploration of inflammatory pathways in GBC.[11] Similarly, another study by Høgdall et al showed serum IL-6 as an independent prognostic
factor in biliary tract cancers, including GBC.[12]
TNF-α is also a pleiotropic cytokine that has been associated with cancer progression.
TNF-α plays a role in cancer promotion via nuclear factor-κb (NF-κb) signaling.[13] TNF-α has been studied in bile, gallbladder cell lines, and gallbladder tissues.[14]
[15] Chaturmohta et al studied the messenger ribonucleic acid (mRNA) expression of various
cytokines, including IL-6 and TNF-α in GBC.[16] They reported an association of increased expression of TNF-α, IL1-β, IL-8, and
VEGF with stages and grades of GBC, whereas IL-6 mRNA failed to show any association.
According to previous studies, IL-6 and TNF-α are the most common inflammatory mediators
in the tumor microenvironment.[17]
[18]
[19] There is a paucity of studies on the evaluation of inflammatory cytokines as potential
serum-based biomarkers in GBC. However, few scattered reports are available in cases
of IL-6,[11]
[12] whereas no such study could be found exploring both IL-6 and TNF-α. The present
study aimed to assess the diagnostic efficacy of serum IL-6 and TNF-α in cases of
GBC, as well as their correlation with different clinicopathological characteristics
and frequently employed serum tumor markers, including CEA, CA125, and CA19-9, which
are utilized to evaluate tumor response and prognosis.
Material and Methods
This prospective case-control study was conducted after obtaining appropriate approval
from Institutional Ethical Committee (Ref. no. 422/IEC/M. Sc/2020). Informed consent
was taken from all the patients. Convenient sampling method was used since this was
a time-bound study. Patients ≤18 years, previously treated for GBC, those with metachronous
and synchronous malignancies, poor performance status, and those who refused to give
consent were excluded. Forty patients aged ≥18 years, with fine needle aspiration
cytology (FNAC) or histopathologically biopsy-proven cases of GBC, were included in
the present study. Contrast-enhanced computed tomography (CECT) of the abdomen and
pelvis was done to determine the disease stage. The staging was done as per the American
Joint Committee on Cancer (AJCC) eighth staging system. Resectability was assessed
based on the radiological investigation. Criteria for unresectability included the
involvement of portal vein, hepatic artery, and direct extension into adjacent organs,
and presence of omental or peritoneal deposits and enlarged aortocaval lymph nodes.
Forty controls were obtained that were matched for age and sex. Healthy controls were
selected from individuals who were ≥18 years with normal liver function tests and
no other comorbidities.
Blood Samples and Cytokines Measurement
Five milliliters of venous blood was collected after taking informed consent in a
plain vacutainer with clot activators (BD Vacutainer, New Jersey, United States).
Serum was separated within 1 hour of blood collection after a centrifugation at 1,500 g for 15 minutes. The serum was stored without preservatives at –80°C and then thawed
before testing. Serum IL-6 concentration was determined using commercially available
human IL-6 high-sensitivity enzyme-linked immunosorbent assay (ELISA) kit (Weldon
Biotech, India). The assay employs the quantitative sandwich immunoassay technique
using recombinant human IL-6, with antibodies raised against the recombinant proteins.
The IL-6 ELISA assay has a performance sensitivity of 0.81 pg/mL.
Serum TNF-α concentration was determined using the commercially available ELISA US
(Ultra-Sensitive) kit supplied by Invitrogen (Waltham, Massachusetts, United States).
The TNF-α assay had a performance sensitivity of less than 0.50 pg/mL, and the minimum
detectable level was 0.09 pg/mL. Optical density was measured using a microtiter plate
reader (BioTek Eon High-Performance Microplate Spectrophotometer, United States) at
450 nm. All standards were run in duplicates, and a standard curve was plotted.
Serum CA19-9, CA125, and CEA levels were investigated by chemiluminescence on ADVIA
Centaur XP system (Siemens Healthineers, Erlangen, Germany). The samples were run
only after a satisfactory level of performance by two levels of internal quality controls
(low and high) as per the manufacturer's instruction. The cutoff levels taken for
CA19-9, CA125, and CEA were 30.9 IU/mL, 30.2 units/mL, and 5 ng/mL, respectively,
as per the specific kits used for analysis.
Statistical Analyses
Baseline characteristics and patient clinical data were expressed in percentages.
Serum IL-6, TNF-α, and tumor markers were reported as median and interquartile range.
Kolmogorov–Smirnov test showed the nongaussian distribution of the values. Receiver
operating characteristics (ROC) curve analysis was performed to determine the clinical
utility of serum IL-6 and TNF-α in identifying GBC patients and to determine the sensitivity,
specificity, and optimal cutoff value of serum IL-6 and TNF-α in GBC patients. Bivariate
Mann–Whitney U test was used to compare serum IL-6 and TNF-α levels in GBC patients and control
groups and also in reference to metastasis, gallstones, resectability, lymph node
involvement, stages, and liver infiltration. Logistic regression was performed to
evaluate the association between clinicopathological characteristics and GBC cases.
Spearman's correlation test was used to find the correlation between serum IL-6 and
TNF-α with serum tumor markers. A probability (p) value less than 0.05 was considered statistically significant. Statistical analysis
was performed using IBM SPSS version 25.0 (IBM Corporation, Armonk, New York, United
States).
Result
A total of 80 samples were enrolled in the study, out of which 40 were GBC patients.
The mean age of the GBC patient group was 53.6 ± 12.42 years. In all, 80% of GBC patients
were females. Abdominal pain (87.5%) was the most common presenting symptom. Most
of the patients were presented at an advanced stage, with 65% in stage IV cancer.
In total, 75% of the patients were associated with gallstones, and 80% had liver infiltration.
Eighty percent of the patients had an unresectable disease and were subjected to palliative
chemotherapy. The healthy control group composed of individuals who were matched in
terms of age and sex, exhibited normal liver function test results, and did not present
with any notable clinical characteristics. The clinicopathological characteristics
of GBC cases are summarized in [Table 1].
Table 1
Clinicopathological characteristics of GBC patients
Variables
|
Frequency (N = 40)
|
Values (%)
|
Gender
|
Male
|
8
|
20.0
|
Female
|
32
|
80.0
|
Personal history
|
Smoking
|
12
|
30.0
|
Alcohol
|
5
|
12.5
|
Clinical presentation
|
Abdominal pain
|
35
|
87.5
|
Jaundice
|
18
|
45.0
|
Fever
|
18
|
45.0
|
Palpable mass
|
23
|
57.5
|
Gallstones
|
30
|
75.0
|
Gallbladder mass
|
36
|
90.0
|
Lymph nodes involvement
|
N0
|
7
|
17.5
|
N1
|
9
|
22.5
|
N2
|
24
|
60.0
|
Metastasis
|
M0
|
15
|
37.5
|
M1
|
25
|
62.5
|
Tumor staging groups
|
I
|
Early stage
|
6
|
15.0%
|
II
|
2
|
5.0%
|
III
|
Late stage
|
7
|
17.5%
|
IV
|
25
|
62.5%
|
Liver infiltration
|
32
|
80.0
|
Resectability
|
Yes
|
8
|
20.0
|
No
|
32
|
80.0
|
Serum Levels of Cytokines in Gallbladder Cancer Patients and Healthy Controls
Serum IL-6 was significantly higher (49.13 [14.12–96.55] pg/mL) in GBC cases as compared
to the control group (4.62 [3.44–5.77] pg/mL). However, TNF-α was found to be undetectable
in the control group, so for statistical purposes, all the control group TNF-α values
were taken as 0.09 pg/mL, which was the lower limit of detection of the kit we used.
For the GBC group, only 42.5% of patients showed TNF-α in a detectable range, from
0.09 to 15.81 pg/mL. Out of the detectable patients, only five showed TNF-α at a higher
level ([Fig. 1], [Table 2]).
Fig. 1 Box plots demonstrating (A) serum IL-6 (values in pg/mL) and (B) TNF-α (values in pg/mL) in gallbladder carcinoma cases and healthy controls. IL-6,
interleukin-6; TNF-α, tumor necrosis factor-alpha.
Table 2
Serum levels of IL-6 and TNF-α in GBC patients and healthy controls
Inflammatory marker
|
n
|
Z value
|
p-value
|
IL-6 (pg/mL)
|
GBC
|
40
|
–6.09
|
0.0001[a]
|
Controls
|
40
|
TNF-α (pg/mL)
|
GBC
|
40
|
–4.57
|
0.0001[a]
|
Controls
|
40
|
Abbreviations: GBC, gallbladder cancer; IL-6, interleukin-6; TNF-α, tumor necrosis
factor-alpha.
a Significant at the 0.05 level.
To determine the clinical utility of serum IL-6 and TNF-α, the ROC curve was plotted
([Fig. 2]). ROC curve analysis revealed an area under curve (AUC) for serum IL-6 of 0.89 with
a sensitivity of 82.5% and specificity of 97.5% at an optimal cutoff value of 13.49
pg/mL as determined by Youden's index. AUC for TNF-α was found to be 0.71 with a sensitivity
and specificity of 40.0 and 100%, respectively, and a cutoff value of 0.24 pg/mL,
indicating the high discriminatory potential of these cytokines in identifying GBC
patients ([Table 3]).
Fig. 2 ROC curves of serum IL-6 and TNF-α for the diagnosis of gallbladder cancer. IL-6,
interleukin-6; receiver operating characteristics; TNF-α, tumor necrosis factor-alpha.
Table 3
ROC curve analysis for serum IL-6 and TNF-α
|
AUC
|
Standard error
|
95% confidence interval
|
Cutoff value (pg/mL)
|
Sensitivity (%)
|
Specificity (%)
|
Lower bound
|
Upper bound
|
|
|
|
IL-6
|
0.89
|
0.04
|
0.81
|
0.97
|
13.49
|
82.5
|
97.5
|
TNF-α
|
0.71
|
0.05
|
0.59
|
0.82
|
0.245
|
40.0
|
100
|
Abbreviations: AUC, area under the curve; IL-6, interleukin-6; ROC, receiver operating
characteristics; TNF-α, tumor necrosis factor-alpha.
Comparison of Serum IL-6 and TNF-α with Clinicopathological Characteristics
The difference in serum IL-6 levels between the GBC resectable and nonresectable groups
was statistically significant (p < 0.05). However, no statistically significant difference was observed for IL-6 levels
with respect to metastasis, gallstones, lymph node involvement, or TNM (tumor size, node involvement, and metastasis status) stage in GBC patients. Serum TNF-α levels do not significantly vary
among the different clinicopathological characteristics of GBC patients ([Table 4]). Both serum IL-6 and TNF-α were not found to be associated with age, gender, gallstones,
total bilirubin, smoking, and alcoholism by univariate and multivariate logistic regression
analyses.
Table 4
Comparison of serum IL-6 and TNF-α with clinicopathological characteristics
Clinicopathological characteristics
|
IL-6
|
TNF-α
|
Median (IQR)
|
p-value
|
Median (IQR)
|
p-value
|
Metastasis
|
Yes
|
56.71 (17.80–96.54)
|
0.31
|
0.24 (0.09–7.96)
|
0.23
|
No
|
47.55 (4.76–108.12)
|
0.09 (0.09–1.50)
|
Gallstones
|
Yes
|
47.84 (14.27–88.98)
|
0.50
|
0.09 (0.09–3.74)
|
0.19
|
No
|
65.66 (8.94–147.19)
|
0.29 (0.09–11.36)
|
Resectability
|
Yes
|
5.12 (2.97–70.90)
|
0.01[a]
|
0.09 (0.09–9.02)
|
0.59
|
No
|
54.30 (20.81–117.19)
|
0.09 (0.09–4.11)
|
Lymph node involvement
|
Yes
|
51.89 (17.80–114.91)
|
0.06
|
0.09 (0.09–5.65)
|
0.98
|
No
|
5.49 (3.78–66.414)
|
0.09 (0.09–3.56)
|
TNM stage
|
Early
|
20.85 (4.02–74.38)
|
0.08
|
0.09 (0.09–9.89)
|
0.88
|
Late
|
54.30 (16.99–117.19)
|
0.09 (0.09–3.59)
|
Liver infiltration
|
Yes
|
50.01 (14.86–117.19)
|
0.27
|
0.09 (0.09–1.34)
|
0.13
|
No
|
44.31 (4.02–76.03)
|
1.90 (0.09–11.53)
|
Abbreviations: IL-6, interleukin-6; IQR, interquartile range; TNF-α, tumor necrosis
factor-alpha; TNM, tumor size, node involvement, and metastasis status.
a Significant at the 0.05 level.
Correlation of IL-6 and TNF-α with Serum Tumor Markers
A significant positive correlation was found between IL-6 and CA125 (p = 0.028, r = 0.348), while there was no significant correlation between TNF-α and tumor markers
([Table 5]).
Table 5
Correlation of serum IL-6 and TNF-α with CEA, CA125, and CA19-9
Tumor markers
|
Median (IQR)
|
(N = 40)
|
IL-6
|
TNF-α
|
CEA (ng/mL)
|
3.97 (2.42–26.86)
|
Correlation coefficient
|
0.29
|
0.18
|
p-value
|
0.06
|
0.25
|
CA125 (units/mL)
|
67.05 (24.5–214.4)
|
Correlation coefficient
|
0.34
|
–0.06
|
p-value
|
0.02[a]
|
0.70
|
CA19-9 (IU/mL)
|
84.21 (14.58–1,200)
|
Correlation coefficient
|
0.24
|
0.05
|
p-value
|
0.12
|
0.73
|
a Significant at the 0.05 level.
Evaluation of Risk Factors in GBC Patients
Univariate and multivariate logistic regression analyses revealed age, gender, gallstones,
total bilirubin, smoking, and alcoholism were not risk factors for GBC ([Table 6]).
Table 6
Univariate and multivariate logistic regression analysis for evaluation of risk factors
for gallbladder cancer
Risk factors
|
Univariate
|
Multivariate
|
Z score
|
p-value
|
Z score
|
p-value
|
Age
|
–1.09
|
0.275
|
–0.996
|
0.31
|
Gender
|
0.413
|
0.679
|
0.623
|
0.62
|
Gallstones
|
–0.706
|
0.48
|
0.488
|
0.48
|
Alkaline phosphatase
|
0.974
|
0.33
|
0.270
|
0.27
|
Total bilirubin
|
–0.045
|
0.964
|
0.075
|
0.94
|
Smoking
|
0.962
|
0.335
|
1.378
|
0.168
|
Alcoholism
|
0.006
|
0.995
|
0.006
|
0.995
|
Discussion
GBC originates from the gallbladder epithelia, which are of highly malignant potential
and the most lethal tumor among different biliary tract cancers worldwide.[1] GBC, owing to its high incidence, late-stage diagnosis, and aggressive tumor behavior
with high morbidity and mortality, deserves extensive clinical research, which directs
toward its early detection. The link between GBC and inflammation is well established,
and the key endogenous promoters include inflammatory cytokines such as IL-1β, IL-6,
and TNF-α, which are the major players in cancer-related inflammation.[20] The pathogenesis and advancement of GBC may be facilitated by regional inflammatory
responses, which entail the discharge of IL-6 and TNF-α. These responses may cause
genetic modifications that promote the survival and proliferation of mutated cells,
impede apoptosis, and stimulate angiogenesis and metastasis.[20] Our study only focused on establishing the relationship between IL-6 and TNF-α with
GBC.
In our investigation, the GBC patients' serum IL-6 and TNF-α levels were found to
be considerably higher than those of healthy controls. As IL-6 and TNF-α are known
to be essential players in cancer-related inflammation, this is consistent with the
findings of earlier studies and supports the link between inflammation and cancer.
Similar research on colorectal cancer by Xu et al revealed a high serum IL-6 level
linked to a poor prognosis for both colorectal cancer overall survival and disease-free
survival, suggesting that serum IL-6 may be a viable biomarker for the detection of
colorectal cancer.[21] In a different study on prostate cancer, Michalaki et al found significantly higher
levels of IL-6 and TNF-α in metastatic diseases than in local illness.[7]
ROC curve was generated to determine the diagnostic efficacy or utility of IL-6 and
TNF-α in GBC patients. At an ideal cutoff value of 13.49 pg/mL, the sensitivity and
specificity of IL-6 were 82.5 and 97.5%, respectively, according to the ROC curve
analysis. The cutoff value for TNF-α was 0.24 pg/mL, and the sensitivity and specificity
were 40.0 and 100%, respectively. No control sample for TNF-α revealed concentrations
above the detectable limit, demonstrating the great specificity of the test. IL-6
was found to be a more sensitive marker than TNF-α. However, it was uncertain if it
had a diagnostic or predictive function.
There was also a significant difference in the levels of serum IL-6 between the groups
of patients with resectable and nonresectable GBC. The median value of the nonresectable
group was significantly higher than that of the resectable group, implying that as
the disease progresses, the release of inflammatory markers also increases. According
to Coussens and Werb's work, inflammation plays a crucial role in the growth of malignancies,
which helps explain our findings.[22] The study's results indicate that the tumor microenvironment, which is primarily
mediated and supported by inflammatory cells, is an essential component of the neoplastic
process because it supports the growth, survival, and migration of cancer cells.[22] The lack of association with other factors such age, gender, gallstones, total bilirubin,
smoking, and alcoholism might be the result of a small sample size or a skewed distribution
of cases since more patients had cancer that was in an advanced stage.
On correlating serum IL-6 and TNF-α with serum tumor markers, we observed a significant
positive correlation between IL-6 and CA125. This may be understood in light of a
prior research by Rajput et al that showed a strong correlation between the increase
of CA125 and IL-6 levels in individuals with benign and malignant ovarian tumors.[23] In benign instances, levels of both biomarkers were almost normal, but the levels
were elevated in malignant cases.[23] However, IL-6 failed to show a correlation with CEA and CA19-9. In our study, TNF-α
fails to show any association with tumor markers.
In our study, age, gender, gallstones, total bilirubin, smoking, and alcoholism were
not found to be risk factors for GBC. This is contrary to earlier studies in which
these were shown to be risk factors. This may be due to the low sample size and noninvolvement
of all stages of GBC stages.
The current study's limitation is the small sample size and restricted geographic
dispersion. The serum levels of IL-6 and TNF-α as indicators of inflammation in other
noncancerous gallbladder inflammatory disorders were not evaluated in this study.
Additionally, fewer patients were in the early stages, so firm conclusions could not
be drawn. Since the bulk of the case group is at an advanced stage, we need to conduct
further research with larger sample sizes that include all GBC stages in order to
comprehend the diagnostic and prognostic roles played by IL-6 and TNF-α.
Conclusion
Serum IL-6 may be utilized to diagnose GBC cases, predict whether a case would be
amenable to resection, and identify instances of GBC in combination with other serum
tumor markers. In contrast, serum TNF-α, despite its high area under the curve, showed
low sensitivity for the detection of GBC. However, further studies encompassing noninflammatory
conditions are required to make a conclusive remark on its utility as a specific biomarker.