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Abstract

BACKGROUND:

Interleukin-33 (IL-33) has been recently discovered as an influential factor in the process of tumor immunity, and is presented in cancer pathogenesis.

OBJECTIVE:

This study aimed to determine the serum levels of IL-33 in patients with benign and malignant Salivary gland tumors (SGTs).

METHODS:

This descriptive cross-sectional study was performed on 47 samples of malignant SGTs including 18 mucoepidermoid carcinoma (MEC), 8 adenoid cystic carcinoma (ADCC), 21 malignant mixed tumor (MMT), and 14 benign pleomorphic adenoma (PA). A control group was considered consisting of 28 healthy subjects. The serum level of IL-33 was measured by using sandwich ELISA method. The data were statistically analyzed through Kruskal-Wallis and Mann-Whitney tests.

RESULTS:

The median concentration of IL-33 was 6.91 in malignant, 5.14 in benign, and 5.01 in healthy cases, with a statistically significant difference ( $P=$ 0.001). The median serum levels of IL-33 increased significantly in ADCC (7.15), MEC (7.03), and MMT (6.91) compared with the control group (5.01) ( $P<$ 0.05). The mean rank of MEC was significantly higher than PA ( $P=$ 0.01). IL-33 concentration was positively and significantly correlated with the tumor stage ( $P=$ 0.02) and tumor size ( $P=$ 0.03).

CONCLUSIONS:

IL-33 could be suggested as a novel biomarker to distinguish different types of SGTs.

Keywords

Salivary gland tumor IL-33 malignant benign biomarker

1. Introduction

Salivary gland tumors (SGTs) represent a heterogeneous group of head and neck neoplasms. They are associated with complex clinicopathological characteristics and distinct biological behaviors. The most prevalent benign and malignant SGTs are pleomorghic adenoma and adenoid cystic carcinoma, respectively. The malignant group consists of diverse tumor entities with a low prevalence. The morphological criteria per se are not enough for diagnosis of the benign or malignant SGTs [1]. Recently, new biomarkers have been suggested for distinguishing the benign and malignant SGTs [2].

A suitable tumor marker of adequate serum level allows earlier diagnosis of tumors. Interleukin-33 (IL-33) is a member of the IL-1 cytokine family that acts as proinflammatory cytokine and is identified as ligand for the ST2 receptor [3]. Full length molecule is activated without maturation for biological carrier behavior [4]. IL-33 is a dual function cytokine, i.e. it plays proinflammatory or protective roles depending on the cellular and cytokine context [5].

Evidence suggests that IL-33, as an epithelial alarmin, has an essential role in response to injury and infection and can activate various immune cells [4]. IL-33 is expressed in the normal salivary glands, skin keratinocytes epithelial cells of the mucosal surface and myofibroblasts, functionally involved in the outcome of some tumor entities such as breast and colorectal cancers, and head and neck squamous cell carcinoma [4, 6, 7, 8]. This marker is positively related with invasion in patients with head and neck cancer [9].

Overexpression of IL-33 has been previously shown in benign and malignant salivary gland tumors and head and neck cancer [2, 10]. Furthermore, elevated IL-33 expression has been observed in MEC, ADCC, ACC [2]. The present study aimed to investigate IL-33 in benign and malignant salivary gland tumors by using ELISA methods and also define its concentration in relation to clinicopathologic features.

2. Materials and method

2.1 Patients

This retrospective cross-sectional study was performed on 61 serum samples of patients with malignant and benign SGTs. The malignant tumors were 18 cases of mucoepidermoid carcinoma (MEC), 8 adenoid cystic carcinoma (ADCC), 21 malignant mixed tumor (MMT). There were 4 acinic cell carcinoma (ACC), 4 squamous cell carcinoma (SCC), 1 adenocarcinoma, 5 malignant neoplasm, 2 salivary duct carcinoma, 1 malignant sarcoma, 1 epithelial myoepithelial carcinoma, 1 undifferentiated carcinoma, 2 pleomorphic adenoma with malignant transformation, and 14 cases of benign pleomorphic adenoma (PA) of the salivary glands, diagnosed in Khalili Hospital, affiliated to Shiraz University of Medical Sciences from 2013 to 2015. The ethics review board project was accomplished under Shiraz University of Medical Sciences Ethics Committee approval No: IR.SUMS.REC.1396.S583. The control group included 28 healthy volunteers without any personal and family history of tumors and no drug treatment. None of them had received chemotherapy, radiotherapy or any other remedial interference before sampling.

The exclusion criteria were the absence of administration of anti-neoplastic chemotherapy, radiotherapy, or involvement in other types of cancer prior to the study. Demographic data were collected including age, sex, characteristics of the lesions type, site, and tumor stage.

2.2 Sampling

After obtaining informed consent forms, 5 ml blood sample was taken from the patients and controls and centrifuged for 10 minutes at 1000 rotations per minute (RPM). The serum was separated and stored in deep freezer at $-$ 80 ${}^{\circ}$ C until determination. IL-33 serum levels were measured with sandwich enzyme-linked immunosorbent assay (ELISA) kit for human IL-33 (Catalog Number D3300, R&D Systems, USA).

2.3 Statistical analysis

Statistical analysis was performed using SPSS software, version 16. Kruskal-Wallis test was used to compare the serum levels of IL-33 among the study groups. ANOVA and Spearman’s correlation coefficient were used to determine the relationship of IL-33 and the stage, tumor size, tumor location, lymph node metastasis, age and sex. A $P$ value of at least $<$ 0.05 was considered to be statistically significant.

Table 1
Basic information of the study groups

Groups	Gender (M:F)	Mean age	Site (Minor:Major)	Metastasis to lymph node	Stage				Tumor size
					1	2	3	4	1	2	3
Control	15:13	47 $\pm$ 14.05	–	–	–	–	–	–	–	–	–
Benign
PA	6:8	44.85 $\pm$ 12.08	1:13	–	–	–	–	–	–	–	–
Malignant
MEC	8:10	52.76 $\pm$ 17.97	0:18	4	3	3	5	7	2	7	9
ADCC	2:6	48.20 $\pm$ 20.14	2:6	2	0	3	2	2	0	3	4
MMT	15:6	45.62 $\pm$ 19.92	0:21	4	4	1	11	1	5	2	10
Total	46:43	49.45 $\pm$ 16.03	3:58	8	7	7	18	10	7	12	23
$P$ -value	0.83	0.57	0.27	0.57					0.02	0.03

MEC: mucoepidermoid carcinoma, ADCC: adenoid cystic carcinoma, MMT: malignant mixed tumor, PA: pleomorphic adenoma; Minor: minor salivary gland, Major: major salivary gland.

3. Results

This research was conducted on 47 samples of malignant tumors, 14 PA, and 28 healthy controls. The patients included 46 males and 43 females, aging 33–65 years old, with the mean age of 49 years. Table 1 represents the baseline data. The tumor size and stage were missing for one case of ADCC and four cases of malignant mixed tumor. The IL-33 serum level was 5.o1 Ng/ml in the control group, 5.14 Ng/ml in the PA, and 6.91 Ng/ml in patients with malignant SGTs (Table 2).

Table 2 shows the mean IL-33 serum concentration in the case and control groups. Kruskal-Wallis test demonstrated that serum IL-33 levels increased significantly in the SGTs compared with the control group ( $P<$ 0.001). Mann-Whitney test revealed that the mean rank of malignant tumors was significantly higher than the benign tumors ( $P=$ 0.024) and control groups ( $P=$ 0.001). Kruskal-Wallis also showed that the IL-33 serum levels were statistically different among the SGT subtypes compared with the control group ( $P<$ 0.002).

Table 2
Compare of IL-33 serum level of control, benign and malignant salivary gland tumor types by Mann-Whitney test

Group		Median (mean rank) i	Median (mean rank) j	$p$ -value
Malignant tumors (i)	Benign tumors (j)	6.91 (55.33)	5.14 (37.61)	0.024
	Control (j)	6.91 (55.33)	5.01 (31.36)	0.001
Benign tumors (i)	Control (j)	5.14 (37.61)	5.01 (31.36)	0.459
MEC (i)	PA (j)	7.03 (59.69)	5.14 (37.61)	0.016*
	ADCC (j)	7.03 (59.69)	7.15 (55.50)	0.702
	MMT (j)	7.03 (59.69)	6.91 (51.52)	0.324
	Control (j)	7.03 (59.69)	5.01 (31.36)	0.001*
ADCC (i)	PA (j)	7.15 (55.50)	5.14 (37.61)	0.118
	MMT (j)	7.15 (55.50)	6.91 (51.52)	0.711
	Control (j)	7.15 (55.50)	5.01 (31.36)	0.020*
PA (i)	MMT (j)	5.14 (37.61)	6.91 (51.52)	0.118
	Control (j)	5.14 (37.61)	5.01 (31.36)	0.459
MMT (i)	Control (j)	6.91 (51.52)	5.01 (31.36)	0.007*

MEC: mucoepidermoid carcinoma, ADCC: adenoid cystic carcinoma, MMT: malignant mixed tumor, PA: pleomorphic adenoma.

Mann-Whitney test revealed that the mean rank of MEC was significantly higher than the PA ( $P=$ 0.016) and control group ( $P=$ 0.001). Likewise, the mean rank of ADCC ( $P=$ 0.002) and malignant mixed tumor ( $P=$ 0.007) was significantly higher than the control groups. No significant difference was observed among other groups (Table 2). The Spearman’s correlation coefficient revealed a positive and significant correlation among the tumor size ( $r=$ 0.356, $P=$ 0.03) and stage ( $r=$ 0.447, $P=$ 0.02) in patients with malignant SGT (Table 1). However, no significant correlation was observed between the IL-33 concentration and tumor location, lymph node metastasis, age and sex ( $P>$ 0.05 in all cases).

4. Discussion

Infiltrations of the immune cells into tumor sites are associated with cytokine production and changes in the cell behavior [11]. Many of these changes are followed by clinical outcome [12]. Cytokines within the tumor microenvironment may affect the tumor cell growth and tumor progression. Increased epithelial expressions of several anti-inflammatory and angiogenesis stimulator cytokines, such as IL-1, have been observed in salivary glandular epithelial cells and salivary gland tumors [2, 4].

Being a member of IL-1 cytokine family, IL-33 acts as proinflammatory cytokine that activates various types of immune cells [3]. However, there is little information about the expression and function of IL-33/ST2 pathway in human tumors [5].

The present study investigated the role of Interleukin-33 in the serum of malignant and benign salivary gland tumors. The results showed that the increase of IL-33 levels in malignant SGTs was significantly higher than that in the benign SGTs and control groups. The results of this study were in line with those of the studies on other tumor types, namely breast, hepatocellular, malignant and benign salivary gland tumors, and head and neck squamous cell carcinoma [2, 8, 13, 14]. Jovanovic et al. investigated the IL-33/ST2 in breast cancer and found that the serum levels of IL33 and sST2 were significantly higher in patients than the healthy subjects. They reported that IL-33 and sST2 could be used as non-invasive diagnostic markers for breast cancer [13]. Rössle et al. reported increased expression of IL-33 in malignant and benign SGTs [2].

Our findings were are also in contrast with the results of some research on colon cancers and lung tumors [7, 12]. O’Donnell et al. observed that the levels of IL-33 and ST-2L decreased in patients with colon cancer compared with the healthy subjects. This reduction was due to the infiltration of the immune cells such as TCD8 and NK cells by the production of chemokine CCL2 [7]. Some studies which are in contrast with our findings even rejected the role of IL-33 in the diagnosis of malignant tumors. This contradiction might be related to differentiation of the tumor, location and the cells causing production of cytokine and the receptor of IL-33 [5, 15].

Controversies exist regarding the pro-inflammatory role of IL-33 as a major pathologic agent in tumor progression and its plausible protective role in reducing the tumor progression. Moreover, the role of IL-33 in salivary gland tumors is still unclear [7]. On the other hand, some studies such as that by Barrera et al. confirmed the protective role of IL-33 in cases such as lung cancer. They observed the cytokine levels of IL-33 in the plasma of non-small-cell in patients with lung cancer compared with the controls [16]. Studies by Naito et al. and O’Donnell et al. found that IL-33 with recruitment of anti-tumor cells such as TC8+ and NK cells to tissues was involved in the survival of patients [7, 17].

The current study noted that the serum level of IL-33 was higher in MEC and ADCC, which are considered as high-grade tumors, than in MMT; however, the difference was not statistically significant. In a study by Rössle et al., the higher expression of IL-33 in tumors like ACC, compared with ADCC, was attributed to the presence of myoepithelial cells in ACC [2]. In colorectal cancer, the expression of IL-33 was observed in tumor tissues, and its serum levels were higher in tumors with low or moderate differentiation than in those with high differentiation. Furthermore, the IL-33 serum levels were higher in cases with colon cancer than those with rectal cancer [10].

Rössle et al. noted that the nuclear IL-33 expression in malignant neoplasms was limited to specific tumors such as acinic cell carcinomas, epithelial-myoepithelial carcinoma, and oncocytic carcinomas. Thus, they suggested that the marker could be used to detect malignant salivary gland subtypes [2]. In our study, the results also showed a significant difference between different SGTs compared with normal salivary glands. In addition, the level of IL-33 cytokine in patients with high-grade tumors such as MEC was higher than in those with PA. Therefore, our results indicated that IL-33 could be suggested as a biomarker for prognosis of the salivary gland tumor subtypes.

Based on the present findings, the tumor stage and size were directly correlated with IL-33 concentration in patients with malignant SGT. These results are similar to what was found by Sun et al. who measured the serum levels of IL-33 in patients with gastric cancer [18]. Xie et al. reported that the IL-33 expression was directly related with the age and sex in patients with colorectal cancers [10]. Musolino et al. demonstrated the IL-33 plasma levels to be inversely associated with the tumor stage in multiple myeloma [19]. On the other hand, the direct relationship between IL-33 expression levels and the patients’ long-term survival shows that IL-33 is a component of inflammatory cytokines that may play a role in immune defense against tumors. According to previous findings, IL-33 increases the TC8+ and NK cell in tumor sites, which consequently leads to elimination of tumors [20].

5. Conclusions

In the present study, the IL-33 serum level increased in patients with different malignant SGTs such as ADCC, MEC and MMT compared to those with benign SGTs and healthy subjects. Moreover, a positive correlation was detected between the IL-33 concentration and the tumor size and stage. Although the mechanism of IL-33 was not investigated in this study, it seems that the increase in IL-33 serum levels could be associated with development of Th2immune response, production of IL-4 and IL-5 cytokines, which prevents the development of Th1 cells and promotes the regulatory T-cells [5]. Consequently, Interleukin-33 promotes the growth of the salivary gland tumors. Yet, further research is required to confirm the role of IL-33 in infiltration of the immune cells in patients with salivary gland tumors. Besides, more similar studies with larger sample size and different populations are needed to confirm its representative role.

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Investigation of IL-33 serum levels in patients with benign and malignant salivary gland tumors

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and method

2.1 Patients

2.2 Sampling

2.3 Statistical analysis

Table 1 Basic information of the study groups

Table 2 Compare of IL-33 serum level of control, benign and malignant salivary gland tumor types by Mann-Whitney test

5. Conclusions

References

Table 1
Basic information of the study groups

Table 2
Compare of IL-33 serum level of control, benign and malignant salivary gland tumor types by Mann-Whitney test