Interleukin-8: A Potential Marker for Differentiating Papillary Thyroid Cancer

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International Journal of Research and Review
Vol.7; Issue: 4; April 2020
Website: www.ijrrjournal.com
Original Research Article E-ISSN: 2349-9788; P-ISSN: 2454-2237
International Journal of Research and Review (ijrrjournal.com) 369
Vol.7; Issue: 4; April 2020
Interleukin-8: A Potential Marker for
Differentiating Papillary Thyroid Cancer
Toral Kobawala1, Kinjal Gajjar1, Nandita Ghosh2
1Research Assistant, 2Assistant Professor and Head,
Tumor Biology Lab, Department of Cancer Biology, The Gujarat Cancer & Research Institute, Asarwa,
Ahmedabad-380016.
Corresponding Author: Nandita Ghosh
ABSTRACT
Background: Thyroid disorders represent
relatively common medical conditions
worldwide. Although better managed, it is often
difficult to distinguish benign and thyroid
cancer cells. To avoid inappropriate treatment
decisions based on unconvincing results, it is
inevitable to understand molecular mechanisms
underlying thyroid carcinogenesis. Cytokines
have a key role in an intricate relationship
between inflammation and cancer. IL-8 is one
such potent inflammatory mediator recognized
to modulate proliferation, invasion and
migration of tumor cells. Hence it was aimed to
study expression of IL-8 in benign and thyroid
cancer patients.
Methods: Circulating IL-8 levels (by ELISA)
and tumoral IL-8 expression (by
Immunohistochemistry) was studied in total 240
subjects: 67 healthy individuals, 67 benign
thyroid diseases patients and 106 thyroid cancer
patients with majority being diagnosed as
papillary thyroid cancer (PTC: N=83).
Results: Circulating IL-8 was significantly
elevated in benign and thyroid cancer as
compared to controls. Both circulating and
tumoral IL-8 expression were significantly
higher in PTC as compared to benign thyroid
diseases. Moreover, IL-8 expression was
significantly associated with aggressive tumor
characteristics of PTC patients.
Conclusion: IL-8 could be a potential marker
for differentiating patients with benign thyroid
diseases and cancer. IL-8 overexpression was
not suppressed by Suppressors of cytokine
signalling (SOCS) proteins and may induce
expression of adhesion molecules: VCAM-1 and
L-Selectin and thereby increase thyroid cancer
progression. Further, significant association of
IL-8 expression with shorter overall survival in
PTC patients treated with surgery alone,
suggests that conventional treatment strategies
may be improved by additionally targeting IL-8
signalling in such patients.
Key words: Thyroid cancer, IL-8, SOCS,
Immunohistochemistry, ELISA, Benign thyroid
diseases
INTRODUCTION
- an endocrine gland plays
important role in regulating metabolism and
helps maintain body temperature, heart rate
and blood pressure. Thus, any problem that
occurs in the thyroid gland, affects every
cell of the body. Thyroid disorders represent
relatively common medical conditions
ranging from benign goitre to carcinoma.
Although its incidence is lower, thyroid
cancer represents the most frequent
endocrine malignancy. Despite the fact that
majority of patients with thyroid diseases
are better managed, pathologists often find
it difficult to distinguish benign and cancer
cells. This may result in unconvincing
treatment decisions by clinicians. Hence, it
is important to decipher the molecular
mechanisms underlying thyroid
tumorigenesis, to avoid excessive treatment
to the patients with indolent/low risk tumors
and at the same time, guarantee effective
management to the patients with aggressive
disease.
Inflammation representing one of the
hallmarks of cancer [1] includes the
existence of inflammatory mediators-

Toral Kobawala et.al. Interleukin-8: a potential marker for differentiating papillary thyroid cancer
International Journal of Research and Review (ijrrjournal.com) 370
Vol.7; Issue: 4; April 2020
cytokines. Shedding of cytokines by tumor
cells into the local microenvironment is a
key modulator of tumorigenesis. Overall,
there is an intricate relationship between the
immune system and cancer where cytokines
have a significant role.[2] Cytokines
modulate an antitumoral response in the
tumor microenvironment, but during
chronic inflammation, they can induce
malignant cell transformation based on
balance of pro- and anti- inflammatory type.
IL-8 is a cytokine recognized as
potent neutrophil activator and chemotactic
factor secreted by activated monocytes,
macrophages, fibroblasts, lymphocytes,
neutrophils, endothelial cells and a variety
of normal and malignant epithelial cells.[3, 4]
The increased secretion of IL-8 from tumor
cells can have profound effect on the tumor
microenvironment. IL-8- CXCR1/2 (IL-8
receptors) signalling mediates tumorigenesis
and tumor progression that leads to
subsequent activation of various
pathways.[5, 6] The IL-8 signalling nexus
directly influences the sensitivity of tumour
cells to chemotherapies by altering
pathways associated with apoptosis and
multidrug resistance.[6]
Increased circulating IL-8 levels and
its correlation with tumor burden and
prognosis have been observed in patients
with various malignancies.[7, 8] Moreover,
statistically significant differences in IL-8
levels in patients with thyroid disease and
normal reference group have also been
demonstrated.[9-12] To date, immense
research has been done to identify the role
of IL-8 signalling in human cancers. Given
that high expression of IL-8 is associated
with tumorigenesis and progression of
certain types of tumours, this study
hypothesized that IL-8 may serve as useful
biomarker in screening and evaluating
prognosis in thyroid cancer patients as well.
Hence it was aimed to study its expression
in benign and thyroid cancer patients and
thereby explore its role as a potential marker
for differentiation and prognosis in thyroid
carcinoma patients.
MATERIALS AND METHODS
Subjects
Total 240 subjects were included, of
which 67 were healthy individuals, 67 were
patients with benign thyroid diseases and
106 were thyroid cancer patients (PTC:
N=83, follicular thyroid cancer (FTC): N=6,
medullary thyroid cancer (MTC): N=9 and
anaplastic thyroid cancer (ATC): N=8).[13-16]
None of the subjects had any history of
autoimmune disease, did not receive any
pre-treatment and they were not on any
immunosuppressive or immunomodulant
drugs. Only 45/67 patients with benign
thyroid diseases that were suspicious to be
malignant were operated at our institute.
This study was approved by
Institutional Scientific and Ethical
Committees and informed consent was
obtained from all subjects prior to sample
collection. Except 4/8 ATC patients who
were not resectable; all thyroid cancer
patients underwent surgery at Department of
Surgical Oncology of our institute. Only
PTC patients were further considered for the
correlation analysis (the number of patients
with other three types of thyroid cancer
were very low for comparative statistical
analysis). Treatment strategies were decided
by the clinicians of the institute. It included
either surgery or surgery followed by
radioiodine ablation (RIA) therapy or
surgery followed by RIA therapy and
radiotherapy both. Clinical and
histopathological details of the patients were
noted from the case files maintained at the
Medical record department of the institute.
Histological classification of the tumors was
in accordance with the WHO classification.
The PTC patients were staged according to
the AJCC/UICC TNM staging system and
were accordingly grouped into younger
      
groups. Clinicopathological characteristics
of PTC patients are depicted in Table 1.[13-
16] Follow up details of PTC patients were
noted for a period of 4 years or until death
within that period. Complete follow-up
details were obtained in 92% (76/83) of
PTC patients and hence were included for

Toral Kobawala et.al. Interleukin-8: a potential marker for differentiating papillary thyroid cancer
International Journal of Research and Review (ijrrjournal.com) 371
Vol.7; Issue: 4; April 2020
overall survival (OS) analysis. Nine percent
(7/76) of these patients had persistent
disease and thus were excluded from disease
free survival (DFS) analysis. Hence, 69/76
PTC patients were included for DFS
analysis.
Table 1: Clinicopathological characteristics of PTC patients
Characteristics
N (%)
Characteristics
N (%)
Age
Bilaterality
<45 years

41 (49)
42 (51)
Unilateral
Bilateral
61 (74)
22 (26)
Gender
Haemorrhagic area
Female
Male
56 (68)
27 (32)
Absent
Present
72 (87)
11 (13)
Tumour size
Necrosis
T1 (N=16)+T2 (N=22)
T3 (N=30)+T4 (N=15)
38 (46)
45 (54)
Absent
Present
67 (81)
16 (19)
Nodal status
Calcification
Absent
Present
30 (36)
53 (64)
Absent
Present
32 (39)
51 (61)
Metastasis
Extrathyroidal extension
Absent
Present
73 (88)
10 (12)
Absent
Present
52 (63)
31 (37)
Stage
Fibrosis
Early [Stage I (N=37) + Stage II (N=12)]
Advanced [Stage III (N=11)+ Stage IV(N=23)]
49 (59)
34 (41)
Absent
Present
61 (74)
22 (26)
Lymphatic permeation
Inflammation
Absent
Present
67 (81)
16 (19)
Absent
Present
46 (55)
37 (45)
Vascular permeation
Differentiation
Absent
Present
74 (89)
09 (11)
Well
Moderate/ Poor
76 (92)
07 (08)
Capsular Invasion
Multifocality
Absent
Present
55 (66)
28 (34)
Absent
Present
64 (77)
19 (23)
Encapsulation
Residual Disease
Well encapsulated
Partially/Not encapsulated
76 (92)
07 (08)
Absent
Present
24 (29)
59 (71)
Treatment
Surgery
29 (35)
Surgery + RIA and/RT
54 (65)
Surgery + RIA
50 (60)
Surgery + RIA +RT
04 (05)
Disease Status
Recurrence/Distant Metastasis (N=69)
Alive/Dead (N=76)
Absent
62 (90)
Alive
68 (89)
Present
07 (10)
Dead
08 (11)
Recurrence
3 (4)
Distant metastasis
4 (6)
Bone
1 (1.5)
Lung
2 (3.0)
Bone + Lung
1 (1.5)
Enzyme Immunoassay (EIA) for
circulating IL-8:
Pretherapeutic blood samples were
collected from all subjects, and sera were
separated and stored at 80⁰C until analysis.
Circulating levels of IL-8 were estimated
from the serum samples using commercially
available kit (EIA IL-8: Immunotech-
   
The unknown concentrations were
determined through Graph pad prism 5
software.
Immunohistochemistry (IHC) for tumoral
protein expression of IL-8:
Formalin fixed paraffin embedded
tissue blocks of the patients were retrieved
from Histopathology department. Four
micron thick sections were taken and
mounted on aminopropyl triethoxy silane
(APES) coated glass slides.
Immunohistochemical staining was
performed using primary mouse monoclonal
IL-8 antibody (R&D Systems-MAB208;
dilution-1:50) and MACH4 Universal HRP-
Polymer Detection System (Biocare
    

Toral Kobawala et.al. Interleukin-8: a potential marker for differentiating papillary thyroid cancer
International Journal of Research and Review (ijrrjournal.com) 372
Vol.7; Issue: 4; April 2020
recommendations. Briefly, the
Immunohistochemical staining procedure
and semiquantitative scoring of the stained
sections were performed as described
earlier.[13]
Statistical analysis
Statistical Package for Social
Sciences (SPSS) version 16 (SPSS Inc,
USA) was used to analyse the data. IL-8
levels between two groups were assessed by
Independent Samples T-test. Discriminating
efficacy of IL-8 was determined by
   
curves. Tumoral protein expressions in
benign and carcinoma patients and
association with clinicopathological
parameters of carcinoma patients were
determined by Two-  
    
used to find correlation between two
parameters. Univariate survival analysis for
DFS and OS was evaluated using Kaplan-

0.05 were considered significant.
RESULTS
Circulating levels of IL-8
Circulating IL-8 levels in different
subjects are depicted in Table 2. In patients
with benign thyroid diseases (N=67), the
circulating IL-8 levels were significantly
higher as compared to that in healthy
individuals (P=0.003). The total benign
thyroid patients were further grouped as
patients with goitre (N=45) and
autoimmune diseases (N=22). It was
observed that the patients with goitre had
significantly higher circulating levels of IL-
8 than the healthy individuals (P<0.001).
Further, patients with autoimmune diseases,
when sub grouped into Hashimoto’s
thyroiditis (N=9) and Graves’ disease
(N=13), it was noted that as compared to the
healthy individuals, the circulating levels of
IL-8 were significantly higher in both the
subgroups of patients- 
    
(P<0.001). In Total thyroid carcinoma
patients (N=106), the circulating levels of
IL-8 were significantly higher than the
healthy individuals (P<0.001). These
thyroid carcinoma patients were further sub
grouped according to their histological
subtypes as PTC, FTC, MTC and ATC. It
was observed that in all these sub groups of
thyroid carcinoma patients, the circulating
levels of IL-8 were predominantly higher
than the healthy individuals (PTC: P<0.001,
FTC: P<0.001, MTC: P<0.001 and ATC:
P<0.001).
Table 2: Significance of circulating levels of IL-8
Subjects
IL-8
M ± SE (pg/ml)
P value
Healthy individuals (N=67)
2.76 ± 0.32
Benign thyroid disease (N=67)
145.15 ± 46.72
0.003*
Goitre (N=45)
144.53 ± 37.46
<0.001*
Autoimmune diseases (N=22)

329.70 ± 296.95
0.002*
N=13)
19.53 ± 10.08
<0.001*
Thyroid carcinoma (N=106)
353.97 ± 65.84
<0.001*; 0.023#
Papillary thyroid carcinoma (N=83)
355.37 ± 78.02
<0.001*; 0.031#
Follicular thyroid carcinoma (N=6)
566.72 ± 325.06
<0.001*; 0.023#
Medullary thyroid carcinoma (N=9)
415.09 ± 192.14
<0.001*; 0.066#
Anaplastic thyroid carcinoma (N=8)
111.21 ± 60.92
<0.001*; 0.806#
*Significance of circulating levels of IL-8 in benign and thyroid carcinoma patients as compared to healthy individuals.
#Significance of circulating levels of IL-8 in thyroid carcinoma patients as compared to benign thyroid diseases.
Further, the ROC curves revealed that IL-8 exhibited a good discriminatory efficacy
between healthy individuals and patients with different thyroid diseases (Figure 1A-1H).

Toral Kobawala et.al. Interleukin-8: a potential marker for differentiating papillary thyroid cancer
International Journal of Research and Review (ijrrjournal.com) 373
Vol.7; Issue: 4; April 2020
A
Area Under the Curve
Area
Significance
95% CI
(P)
Lower
Upper
0.828
<0.001
0.752
0.905
B
Area Under the Curve
Area
Significance
(P)
95% CI
Lower
Upper
0.846
<0.001
0.761
0.932
C
Area Under the Curve
Area
Significance
(P)
95% CI
Lower
Upper
0.897
<0.001
0.719
1.000
D
Area Under the Curve
Area
Significance
(P)
95% CI
Lower
Upper
0.719
0.013
0.513
0.924
E
Area Under the Curve
Area
Significance
(P)
95% CI
Lower
Upper
0.882
<0.001
0.821
0.943
F
Area Under the Curve
Area
Significance
(P)
95% CI
Lower
Upper
0.856
0.004
0.596
1.000

Toral Kobawala et.al. Interleukin-8: a potential marker for differentiating papillary thyroid cancer
International Journal of Research and Review (ijrrjournal.com) 374
Vol.7; Issue: 4; April 2020
G
Area Under the Curve
Area
Significance
(P)
95% CI
Lower
Upper
0.990
<0.001
0.969
1.000
H
Area Under the Curve
Area
Significance
(P)
95% CI
Lower
Upper
0.965
<0.001
0.920
1.000
Figure 1: ROC curve for IL-8 in patients with thyroid diseases vs healthy individuals
A. Total benign thyroid disease patients vs Healthy individuals
B. Benign goitre patients vs Healthy individuals
C. Hashimoto thyroidtis patients vs Healthy individuals
D. 
E. PTC patients vs Healthy individuals
F. FTC patients vs Healthy individuals
G. MTC patients vs Healthy individuals
H. ATC patients vs Healthy individuals
Moreover, it was observed that IL-8 levels were significantly higher in thyroid carcinoma
patients, as compared to patients with benign thyroid diseases (IL-8: P=0.023). Further, when
sub grouped, the levels were found to be considerably higher in PTC and FTC patients as
compared to patients with benign thyroid diseases (PTC: P=0.031 and FTC: P=0.023). [Table
2]. In agreement to this, ROC curves also showed that IL-8 could well discriminate
between patients with benign diseases and PTC as well as between benign and FTC patients
[Figure 2A and B].
A
Area Under the Curve
Area
Significance
(P)
95% CI
Lower
Upper
0.611
0.020
0.521
0.701
B
Area Under the Curve
Area
Significance
(P)
95% CI
Lower
Upper
0.757
0.038
0.500
1.000
Figure 2: ROC curve for IL-8 in patients with thyroid cancer vs benign thyroid diseases
A. PTC vs Benign thyroid disease patients
B. FTC vs Benign thyroid disease patients
Tumoral protein expression of IL-8

Toral Kobawala et.al. Interleukin-8: a potential marker for differentiating papillary thyroid cancer
International Journal of Research and Review (ijrrjournal.com) 375
Vol.7; Issue: 4; April 2020
Cytoplasmic and/or nuclear staining was observed for IL-8 (Figure 3). The immunoreactivity
was either focal or scattered. For statistical evaluation, cytoplasmic and nuclear expressions
were scored independently and taken into account separately.
A
B
C
Figure 3: Photomicrographs showing staining for IL-8
A. Cytoplasmic and nuclear staining for IL-8 in benign goitre
B. Cytoplasmic and nuclear staining for IL-8 in PTC
C. Negative control for IL-8 in PTC
Median IRS of IL-8 expression was used as cut-off to divide the patients into low

3 depicts the comparison of IL-8 expression between the patients with benign thyroid
diseases and thyroid carcinomas.
Incidence of cytoplasmic IL-8 immunoreactivity was significantly high in thyroid cancer
2=8.784, r=+0.244, P=0.003). In
PTC patients too, the incidence of cytoplasmic immunoreactivity of IL-8 was found to be
significantl          2=8.474, r=+0.257,
P=0.003). In FTC patients, the cytoplasmic IL-8 expression was higher than benign thyroid
         2=1.624, r=+0.252,
P=0.074).
Table 3: Comparison of cytokine expressions between the patients with benign thyroid diseases and total thyroid carcinoma patients
Cytokine
expression
BTD (N=45)
N (%)
TTC (N=102)
N (%)
PTC (N=83)
N (%)
FTC (N=6)
N (%)
MTC (N=9)
N (%)
ATC (N=9)
N (%)
Cytoplasmic
IL-8
Median IRS- 2
Median IRS- 4
Median IRS- 4
Median IRS- 1.5
Median IRS- 4
Median IRS- 2
Low
37 (82)
58 (57)
47 (57)
3 (50)
5 (56)
3 (75)
High
8 (18)
44 (43)
36 (43)
3 (50)
4 (44)
1 (25)
2=8.784,
r=+0.244,
P=0.003
2=8.474,
r=+0.257,
P=0.003
2=1.624,
r=+0.252,
P=0.074
2=1.736,
r=+0.239,
P=0.188
2=0.000,
r=+0.051,
P=1.000
Nuclear IL-8
Median IRS- 0
Median IRS- 0
Median IRS- 0
Median IRS- 0
Median IRS- 0
Median IRS- 1
Low
38 (84)
90 (88)
75 (90)
5 (83)
8 (89)
2 (50)
High
7 (16)
12 (12)
8 (10)
1 (17)
1 (11)
2 (50)
2=0.399, r-
0.052, P=0.531
2=0.987, r=-
0.088, P=0.324
2=0.000,
r=+0.010,
P=1.000
2=0.000, r=-
0.047, P=1.000
2=1.063,
r=+0.244,
P=0.302
BTD- Benign Thyroid diseases; TTC- Total Thyroid Cancer patients
Correlation of IL-8 with
clinicopathological parameters of PTC
patients
Preponderance of serum IL-8 levels was
observed in male patients (P=0.035). Also
IL-8 levels were observed to be higher in
patients having larger tumor size (P=0.050),
advanced stage disease (P=0.045) and
presence of fibrosis (P=0.005).
Cytoplasmic IL-8 expression was
significantly higher in males 2=4.112,
r=+0.223, P=0.043); in patients with larger
  2=3.970, r=+0.219, P=0.047)
and presence of extrathyroidal extension of
 2=9.006, r=+0.329, P=0.002).
Moreover, a trend of higher cytoplasmic IL-
8 immunoreactivity was evident in patients
showing capsular invasion of tumors
2=3.262, r=+0.198, P=0.072) and in those

Toral Kobawala et.al. Interleukin-8: a potential marker for differentiating papillary thyroid cancer
International Journal of Research and Review (ijrrjournal.com) 376
Vol.7; Issue: 4; April 2020
having tumors in single lobe of the thyroid
 2=3.160, r=-0.195, P=0.077) as
compared to their respective counterparts.
On the other hand, higher nuclear IL-8
expression was predominant in patients with
smaller tumor size than in patients with
   2=4.487, r=-0.273,
P=0.034). Besides this, a trend of higher
nuclear IL-8 immunoreactivity was seen in
PTC patients with presence of distant
 2=3.081, r=+0.255, P=0.079)
and those who had been postoperatively
    2=3.206,
r=+0.239, P=0.073). Apart from these, IL-8
did not show significant correlation with
rest of the clinicopathological parameters
[Table 4].
Table 4: Correlation of IL-8 with clinicopathological parameters of PTC patients
Parameter
Circulating levels
Tumoral protein expression
Cytoplasmic
Nuclear
Mean ± SE
(pg/ml)
P
Low
N (%)
High
N (%)
Low
N (%)
High
N (%)
Gender
Female
241.48 ± 76.66
0.035
36 (64)
20 (36)
r=+0.223
Male
591.58 ± 173.36
11 (41)
16 (59)
P=0.043
Tumour size
Small (T1+T2)
189.64 ± 76.60
0.050
26 (68)
12 (32)
r=+0.219
31 (82)
7 (18)
r=-0.273
Large (T3+T4)
495.31 ± 125.65
21 (47)
24 (53)
P=0.047
44 (98)
1 (2)
P=0.034
Stage
Early (I+II)
225.72 ± 67.74
0.045
Advanced (III+IV)
542.21 ± 159.75
Metastasis
Absent
68 (93)
5 (7)
r=+0.255
Present
7 (70)
3 (30)
P=0.079
Fibrosis
Absent
224.65 ± 47.43
0.005
Present
717.81 ± 251.70
Extrathyroidal extension
Absent
36 (69)
16 (31)
r=+0.329
Present
11 (35)
20 (65)
P=0.002
Capsular Invasion
Absent
35 (64)
20 (36)
r=+0.198
Present
12 (43)
16 (57)
P=0.072
Bilaterality
Unilateral
31 (51)
30 (49)
r=-0.195
Bilateral
16 (73)
6 (27)
P=0.077
Treatment
Surgery
29 (100)
0 (0)
r=+0.239
Surgery + RIA and/RT
46 (85)
8 (15)
P=0.073
r- correlation coefficient
Survival analysis
The median level of IL-8 (34.20 pg/ml) and median IRS score was used as cut-off to
             
respectively. Univariate analysis revealed that neither circulating IL-8 nor the tumoral IL-8
expression was a significant predictor of DFS or OS in PTC patients [Table 5].
Table 5: Univariate survival analysis for DFS and OS in relation to IL-8 expression in PTC patients
DFS (N=69)
OS (N=76)
N
Patients relapsed N (%)
N
Patients died N (%)
Circulating IL-8 levels
Low
35
3 (9)
38
4 (10)
High
34
4 (12)
38
4 (10)
Log rank=0.170, df=1, P=0.680
Log rank=0.001, df=1, P=0.969
IL-8 protein expression
Cytoplasmic IL-8
Low
39
6 (15)
42
4 (9)
High
30
1 (3)
34
4 (12)
Log rank=2.572, df=1, P=0.109
Log rank=0.099, df=1, P=0.753
Nuclear IL-8
Low
62
7 (11)
68
7 (10)
High
7
0 (0)
8
1 (12)
Log rank=0.831, df=1, P=0.362
Log rank=0.056, df=1, P=0.813

Toral Kobawala et.al. Interleukin-8: a potential marker for differentiating papillary thyroid cancer
International Journal of Research and Review (ijrrjournal.com) 377
Vol.7; Issue: 4; April 2020
However, cytoplasmic IL-8 expression was significant predictor of OS in subgroup of
patients treated with surgery alone. In this group, 25% (3/12) patients with high cytoplasmic
IL-8 expression had significantly shorter OS while, all the patients having lower cytoplasmic
IL-8 expressions remained alive (Log rank=4.106, df=1, P=0.043) [Figure 4].
Figure 4: Significantly reduced OS observed in PTC patients treated with surgery alone having high cytoplasmic IL-8 expression as
compared to those with low cytoplasmic IL-8 expression
Besides this, IL-8 expression when correlated with previous results on the expression
of adhesion molecules (L-Selectin and VCAM-1)[14] and with SOCS proteins (SOCS-1,
SOCS-2 and SOCS-3)[17] in PTC patients, it was observed that cytoplasmic IL-8 expressions
exhibited a significant positive correlation with L-Selectin expression (r=+0.258, P=0.018) as
well as with VCAM-1 (r=+0.437, P<0.001). It also showed significant positive correlation
with the expression of all the three SOCS proteins (cytoplasmic IL-8 vs SOCS-1: r=+0.435,
P<0.001; cytoplasmic IL-8 vs SOCS-2: r=+0.230, P=0.036; cytoplasmic IL-8 vs SOCS-3:
r=+0.336, P=0.002). In addition, nuclear IL-8 expression (r=-0.244, P=0.026) showed a
significant inverse correlation with SOCS-3 immunoexpression [Table 6].
Table 6: Correlation of IL-8 with adhesion molecules (L-Selectin and VCAM-1) and SOCS (SOCS-1, SOCS-2 and SOCS-3) proteins
in primary tumors of PTC patients
Circulating IL-8
Cytoplasmic IL-8 expression
Nuclear IL-8 expression
Circulating L-Selectin
r=+0.165,P=0.135
-
-
Circulating VCAM-1
r=-0.044, P=0.695
-
-
L-Selectin expression
-
r=+0.258, P=0.018
r=-0.062, P=0.579
VCAM-1 expression
-
r=+0.437, P<0.001
r=-0.124, P =0.266
SOCS-1 expression
-
r=+0.435, P<0.001
r=-0.184, P=0.096
SOCS-2 expression
-
r=+0.230, p=0.036
r=-0.066, P=0.551
SOCS-3 expression
-
r=+0.336, p=0.002
r=-0.244, P=0.026
r- correlation coefficient
DISCUSSION
Serum IL-8 levels were significantly
elevated in all patients with thyroid
disorders as compared to healthy
individuals. Its levels were even elevated in
the thyroid cancer patients as compared to
the patients with benign thyroid diseases.
Confirming the results, ROC curves also
revealed that serum IL-8 showed good
efficacy to discriminate between healthy
individuals and patients with different
thyroid diseases as well as between patients
with benign thyroid diseases and thyroid
cancer patients. Contrarily, studies of
Krassas and colleagues found that IL-8
 
    
thyroiditis.[18] However, similar to our
observation, some studies demonstrated
statistically significant differences in IL-8
levels in patients with thyroid disease and
normal reference group.[9-12] Our
preliminary study revealed significant
higher IL-8 levels in benign, autoimmune
and thyroid carcinoma patients, and it was
significantly associated with the advanced
stage disease in PTC patients.[19] Increased
circulating IL-8 levels have also been

Toral Kobawala et.al. Interleukin-8: a potential marker for differentiating papillary thyroid cancer
International Journal of Research and Review (ijrrjournal.com) 378
Vol.7; Issue: 4; April 2020
observed in patients with various other
malignancies.[20-33]
Moreover, high IL-8 levels were
significantly positively correlated with
larger tumor size, advanced stage and
presence of fibrosis in PTC patients. Also
the levels were found to be higher in the
male patients who are more likely to be
associated with aggressive tumor behaviour
as compared to the female patients.
Recently, Sanmamed and colleagues
reported that serum IL-8 levels correlate
with tumor burden and prognosis.[8] In colon
cancer patients, its levels statistically
correlated with tumor stage,[23] and in breast
cancer, circulating IL-8 significantly
increased with tumor size[29] which is
consistent to the present results. IL-8 levels
increased significantly in patients with more
advanced stage in breast and uterine
endometrial cancers.[28, 34]
Similar to present study, de Campos
et al observed IL-8 immunostaining in
cytoplasm and focally in nucleus of
neoplastic cells in breast cancer patients,[35]
while IL-8 protein expression was seen
predominantly in cytoplasm of lung cancer
cells.[36, 37] Jenkins et al had observed a
significant increase in moderate IL-8
    
and an increase in strong staining in
adenocarcinoma tissue compared to adjacent
squamous tissue.[38] Current study also
demonstrated significantly higher
cytoplasmic IL-8 expression in PTC patients
as compared to benign thyroid diseases.
Cytoplasmic IL-8 overexpression was
significantly higher in male patients and was
substantially positively correlated with
larger tumor size and extrathyroidal
extension of tumors, while higher nuclear
expression was associated with smaller
tumor size. This indicates that cytoplasmic
expression might be related to more adverse
tumor characteristics while; nuclear IL-8
immunoreactivity may be indicative of less
hostile tumor behaviour in PTC patients.
In accordance to present study, Chen
et al reported higher IL-8 expression in
pancreatic cancer patients at both circulating
and tumor tissue levels.[39] IL-8 expression
correlated with disease progression in
prostate, breast and ovarian cancers.[40-44]
They observed increase in growth,
proliferation, angiogenesis, adhesion and
invasion with increased IL-8 overexpression
and these effects were decreased on
depletion of endogenous IL-8 expression by
transfecting cells with plasmid encoding for
antisense IL-8.[45]
IL-8 is found to be a prognostic
marker in various human cancers.[46-49] In
present study, higher cytoplasmic IL-8
immunoreactivity was associated with
significantly reduced OS in the PTC patients
who were treated with surgery alone. Thus,
it can be suggested that the patients having
higher IL-8 immunoreactivity may require
more active treatment rather than surgery
alone, for better prognosis.
Moreover, IL-8 expression exhibited
significant positive correlation with the
expression of adhesion molecules in PTC
patients. It can be suggested that, an
increased expression of adhesion molecules
may be the result of neutrophil activation by
inflammatory cytokines like IL-8.[50] In
breast cancer cells, VCAM-1 expression
was induced by cytokoine stimulation and
its up regulation directly correlated with
advanced stage.[51] It has been observed that
addition of exogenous cytokines induced
expression of endothelial adhesion
molecules thereby increasing the adhesive
property of cancer cells. This cell-cell
adhesion leads to clustering of VCAM-1
which in turn activates the PI3K/AKT
signalling that suppresses apoptosis and
promotes survival signal in the tumor
cells.[52]
These results indicate that
production of IL-8 by cancer cells may be
one of the factors leading to expression of
adhesion molecules, which can facilitate
tumor progression through activation of
various signalling pathways and further
substantiate the link between inflammation
and cancer progression.
Further, the expression of SOCS
proteins might be heterogeneously induced

Toral Kobawala et.al. Interleukin-8: a potential marker for differentiating papillary thyroid cancer
International Journal of Research and Review (ijrrjournal.com) 379
Vol.7; Issue: 4; April 2020
by various cytoiknes including IL-8.
Usually, SOCS expression is known to be
stimulated by activation of cytokine
signalling pathway. In turn, when SOCS are
overexpressed, they tend to inhibit cytokine
induced signal transduction in a negative
feedback manner.[53, 54] Hence overall,
cancer cells are sustained by several
cytokines within the tumor
microenvironment, which lead to activation
of pathways that support cancer cell growth
and survival. Expression of SOCS proteins
may be a consequence of this.[54] Thus, in
the present study, significant positive
correlation of IL-8 with the SOCS proteins,
is indicative of a possibility that, there might
be failure of negative regulatory pathways
acting upon the IL-8 induced pathways,
which may overpower the capacity of SOCS
proteins to suppress IL-8 expression and
reduce the activity of downstream
transcriptional factors.
Additionally, the varied effects of
IL-8 signalling upon different cell types
present within the tumor microenvironment
has been suggestive of targeting of CXC-
chemokine signalling including IL-8, in
order to arrest disease progression and assist
in sensitizing tumors to chemotherapeutic
and biological agents. Repertaxin, is the
CXCR1/2 inhibitor developed to prevent IL-
8-induced injury.[55] Clinical trials are in
progress to determine the safety and
efficacy of repertaxin in combination with
docetaxel chemotherapy in patients with
advanced breast cancer.[56-58] Furthermore,
evidence for IL8-CXCR1/2 axis in CSC has
been reported by independent studies and
offers a potential therapeutic target.[59-63]
Currently, randomized, double blind phase 2
clinical trials aimed at testing the effective
targeting of CSC through this axis are in
progress.[56-58, 63]
CONCLUSION
IL-8 is not only produced by
immune cells but also by the follicular cells
of thyroid gland. Its overexpression at both
circulating and tumor tissue levels may
indicate an excessive production by tumor
cells in an inflammatory microenvironment
and subsequent release into the circulation.
Overall, IL-8 has a role as a differentiating
marker in patients with various thyroid
diseases and in advancement of thyroid
cancer. Further, IL-8 expression is not
suppressed by the SOCS proteins and it may
have a role in inducing the expression of
adhesion molecules like VCAM-1 and L-
Selectin and thereby increasing cancer cell
proliferation and progression in PTC
patients. Moreover, as its expression was
able to predict OS in PTC patients treated
with surgery alone, targeting IL-8 signalling
along with conventional treatment strategies
might be beneficial in such patients.
ACKNOWLEDGMENT
This work was financially supported by Gujarat
Cancer Society (GCS), and it was approved by
the GCRI/GCS ethics committee.
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How to cite this article: Kobawala T, Gajjar K,
Ghosh N. Interleukin-8: A potential marker for
differentiating papillary thyroid cancer.
International Journal of Research and Review.
2020; 7(4): 369-382.
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