Research Article
IL-1β and TNF-Α as a Biomarker of Recurrence in Malignant Eyelid Tumours
Sayeed K1, Kaur A1*, Bhasker SK1 and Pant AB2
1Department of Ophthalmology, King George’s Medical University, India
2Department of Ophthalmology, Indian Institute of Toxicology and Research, India
*Corresponding author: Kaur A, Department of Ophthalmology, King George’s Medical University, 226003, Lucknow, India
Published: 28 Jun, 2016
Cite this article as: Sayeed K, Kaur A, Bhasker SK, Pant AB. IL-1β and TNF-Α as a Biomarker of Recurrence in Malignant Eyelid Tumours. Clin Surg. 2016; 1: 1047.
Abstract
Aim: To establish a correlation between cytokine levels (IL-1β, TNF-α and IL-10) and establishing them as a biomarker of recurrence in malignant eyelid tumours.
Method: Prospective observational cross-sectional case-control study of 38 consecutive cases of
malignant eyelid tumours that underwent surgical treatment over a period of 18 months. 26 age and
sex matched controls with other non-inflammatory, non- neoplastic eyelid disorders. The levels of
the cytokines [IL-1β (Interleukin- 1β), TNF-α (tumour necrosis factor-α), IL-10 (Interleukin-10)]
(in pg/ml) were determined by using ELISA Kit. Data was analyzed statistically.
Result: On comparing the mean cytokine levels of the two groups, t-test revealed significantly
higher levels of IL- 1β (16.39 ± 2.86 vs. 14.62 ± 3.72, t=2.17, p=0.034), TNF-α (19.32 ± 3.47 vs. 16.74
± 4.45, t=2.61, p=0.011) and IL-10 (24.79 ± 5.87 vs. 21.61 ± 4.61, t=2.33, p=0.023) in cases. ANOVA
revealed significantly different levels of IL- 1β (F=7.86, p<0.001), TNF-α (F=7.42, p<0.001) and IL-
10 (F=4.36, p=0.006) between tumour and it’s three adjacent tissues.
Conclusion: Cytokine levels (IL-1β, TNF-α and IL-10) in the adjacent tissues beyond safety margins
were normal and comparable to that in controls suggesting the margins to be tumour free. Highly
significant lower levels of IL-1β and TNF-α in tissues beyond safety margins can be used as an
important predictor for local recurrence of tumour.
Keywords: Malignant eyelid tumour; IL-1β, TNF-α; Recurrence
Introduction
Cytokines, a diverse group of small proteins, are important negative and positive regulators
of cell activity. Ample evidence of their role in the diagnosis and treatment of various systemic
tumours is present. However, specific characterization of cytokines in malignant eyelid tumours has
seldom been done.
IL-1β (Interleukin- 1β) is a crucial mediator of the host inflammatory response in natural
immunity with a proinflammatory effect [1]. Expression of IL-1 has been correlated with various
systemic tumours [2,3].
TNF-α (tumour necrosis factor-α) is a multi-functional cytokine having tumour-promoting as
well as tumour-inhibiting activity in varying tissue micro-environments and have been demonstrated
to promote metastatic behaviour in cancer cells via diverse mechanisms various systemic tumours
[4].
IL-10 (Interleukin-10) is an important immunoregulatory cytokine and has been shown to have
diverse effects regarding it’s influence on cancer. IL-10 has been identified in the serum and tumour
with a negative correlation between circulating levels of IL-10 and prognosis [5-8].
The current study aims at estimating the levels of cytokines, viz. IL-1β, TNF-α and IL-10 in
histopathologically confirmed tissue samples of malignant eyelid tumours in comparison to other
non-inflammatory, non- neoplastic eyelid disorders. Additionally, differences between tumour and
its adjacent tissues beyond safety margins will aide in establishing a correlation between cytokine
levels and tumour recurrence.
Method
Study was conducted according to tenets of declaration of Helsinki after approval from
Institutional Ethics Committee. A prospective observational crosssectional
case-control study, was conducted by recruitment of 38
consecutive cases of malignant eyelid tumours that underwent surgical
treatment over a period of 18 months from August 2014 to January
2016 in the Department of Ophthalmology, King George’s Medical
University, Lucknow, India. 26 patients with non-inflammatory,
non- neoplastic eyelid disorders [tissues from senile eyelid disorders,
ptosis (non-inflammatory)] were recruited as controls. An informed
consent was taken from all the patients.
Excised tissues (4 samples were obtained from each case –1 from
tumour mass and 3 samples from adjacent tissue including medial,
base, and lateral) were collected in sterile Dulbecco’s Modified Eagle’s
Medium (DMEM) supplemented with 10% Fetal Bovine Serum
(FBS) and antibiotic- antimycotic solution (Gibco BRL, USA), and
transported to In Vitro Toxicology Laboratory, Indian Institute
of Toxicology Research, Lucknow, India, at a temperature of -4°C
immediately and were preserved in deep freezer at -80°C till further
processing. The levels of the cytokines (IL-1β, TNF-α, IL-10) in the
tissue protein samples (100 μl tissue supernatant) was determined
by using commercially available “Ready-SET-Go! ELISA Kit” (Sigma
Aldrich Chemie GmbH, Buchs, St. Gallen) in triplicate wells. The
analyses of the plates were done at 450 nm using Multiwell microplate
reader (Synergy HT, Bio-Tek, USA). Control samples were also
analyzed by identical procedure.
Expression of the results was done as mean (SEM) and data were
summarized as standard deviation (Mean ± SD) from the values
obtained from at least three independent experiments, in each of
which triplicate samples were used. Comparison of the groups was
done by independent Student’s ‘t’test, ANOVA and Tukey post hoc
test. P-value less than 0.05 were considered statistically significant.
SPSS software (Windows version 17.0) was used for statistical
analyses.
Table 1
Table 2
Results
The present study recruited surgically excised tissues (1 tumour
tissue and 3 adjacent tissues) of 38 patients of malignant eyelid
tumour of either sex as cases and 26 age and sex matched tissue
samples as controls. Subjects of two groups were demographically
matched and comparable (Table 1).
In controls, the tissues included were rectus muscle (69.2%),
tarsus (26.9%) and muller’s muscle (3.8%) and in cases, sebaceous
gland carcinoma (47.36%), basal cell carcinoma (26.3%), squamous
cell carcinoma (18.4%) and malignant melanoma (7.89%).
Comparison of cytokine levels of cases and controls
The cytokine levels of cases and controls are summarized in Table
2. Student’s t-test for the mean cytokine levels (in pg/ml) of the two
groups, revealed significantly higher levels of IL- 1β (16.39 ± 2.86 vs.
14.62 ± 3.72, t=2.17, p=0.034), TNF-α (19.32 ± 3.47 vs. 16.74 ± 4.45,
t=2.61, p=0.011) and IL-10 (24.79 ± 5.87 vs. 21.61 ± 4.61, t=2.33,
p=0.023) in cases as compared to controls. No definite correlation
was seen amongst the three cytokines.
Comparison of cytokine levels of tumour and it’s adjacent tissues
On comparing the mean cytokine levels of four groups, ANOVA
revealed significantly different levels of IL- 1β (F=7.86, p <0.001),
TNF-α (F=7.42, p <0.001) and IL-10 (F=4.36, p=0.006) among the
groups (Table 2 and 3). Further, Tukey post hoc test showed that the
mean level of IL- 1β, TNF-α and IL-10 also lowered significantly
in medial, base and lateral as compared to tumour tissue [Cases vs.
Medial: IL- 1β<0.001 , TNF-α=0.001 and IL-10=0.005; Cases vs. Base:
IL- 1β<0.001, TNF-α<0.001 and IL-10=0.083; Cases vs. Lateral: IL-
1β=0.001, TNF-α=0.001 and IL-10=0.022]. This lowering of cytokine
level was highly significant in case of IL- 1β and TNF-α (p <0.001) but
in case of IL-10, p=0.006
Table 3
Discussion
The current study analyses the cytokines, viz IL-1β, TNF-α and
IL-10 in malignant tumours of eyelid, it’s adjacent tissue beyond the
safety margins and compares them with the local milieu of periocular
tissues. It attempts to study their role as a prognostic marker in eyelid
tumours.
IL-1β is a pro-inflammatory pro- tumour cytokine. The levels of
IL-1β were significantly raised in tumour tissue. Expression of IL-
1β has been correlated with tumour cell proliferation, in previous
studies [9,10]. This leads to the possibility that it may directly increase
proliferation of tumour cells.
In our study, the levels of TNF-α was demonstrated to be
significantly higher in cases than in control tissues. TNF-α is the most
widely studied cytokine and has been demonstrated in various studies
to be a multi-functional cytokine with different actions in different
tissues [11,12]. This suggests its role in tumourigenesis of eyelid
tumours.
IL-10 has a complex biological activity in tumours and has
diverse effects regarding its influence on cancer. In our study, its
levels in cases were seen to be raised significantly in comparison to
controls which is in accordance with the previous studies in which it
was suggested to serve as a tumour growth factor [13-15].
Cytokines levels in tissues adjacent to the cut margin beyond
safety margins were less than that in tumour tissues in the study.
It can be inferred that the surgical margins were tumour free on all
the three adjacent sides beyond 5mm of the surgical safety margins
of excision. These levels were similar to the control values (p>0.05).
There was no significant variation in cytokine levels among the three
adjacent sides (p>0.05), indicating that the three adjacent tissues
were without tumour invasion. This indirectly supports the surgical
safe margin concept [16-18]. The difference in levels were highly
significant in case of TNF- α and IL-1β (p <0.001). This decrease in
cytokine levels were observed on all the three adjacent sides in case
of TNF-α, IL-1β with highly significant lowered levels but not in IL-
10. Thus, TNF-α and IL-1β are more important prognostic markers
in case of recurrence. Also we can use the side with highest TNF-α
and IL-1β levels for vigorous follow-ups to look for any evidence of
recurrence.
No recurrence was noted in the study period. The fact that the
peri tumour cytokine levels beyond safety margin were normal can
be used as an indicator of tumour Free State, therefore, be utilized as
a prognostic marker. Similar use of cytokines (IL-6 and TNF-α) as
prognostic markers for prostate cancer was suggested by Michalaki
et al. [19]. (2004) and were correlated directly with the extent of
malignant disease.
Conclusion
The current study on tissue levels of cytokines (IL-1β, TNF-α
and IL-10) in malignant eyelid tumours and it’s adjacent tissues
beyond safety margins concluded that significantly higher levels were
present in the tumour tissues. Cytokines were found to be normal
in the adjacent tissues beyond safety margins and comparable with
controls, thus establishing the margins to be tumour free. This finding
enables us to use IL-1β and TNF-α as an important predictor for local
recurrence of tumour. This observation at the biomolecular level
establishes the surgical excision margin of 5mm as safe.
The prognostic significance of cytokine levels in adjacent tissues
beyond safety margins, will encourage further undertakings directed
towards the development of tumour treatment agents.
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