Serum IL-8 acts as a novel biomarker in the diagnosis of patients with laryngeal cancer


 Background: Cytokines has the capacity to serve as biomarkers and therapy targets in a number of cancers. The aim of this study was to estimate the diagnostic potential of serum IL-8 in laryngeal cancer.Methods: Serum levels of IL-8 in 126 laryngeal cancer patients and 112 healthy controls were detected using enzyme-linked immunosorbent assay (ELISA) analysis. The association of IL-8 with clinicopathological characteristics was analyzed via χ2 test. Additionally, the receiver operating characteristic (ROC) curve was plotted to evaluate the diagnostic value of serum IL-8 in laryngeal cancer by calculating the area under ROC curve (AUC).Results: Serum IL-8 was significantly increased in laryngeal cancer patients, and its increased expression was positively associated with T classification (P=0.031), clinical stage (P=0.021) and lymph node metastasis (P=0.023). ROC curve suggested that serum IL-8 had a high value in differentiating laryngeal cancer patients from healthy individuals with a AUC value of 0.859 (95%CI: 0.812-0.906) combing with a sensitivity of 86.5% and a specificity of 72.3%.Conclusion: Serum IL-8 was significantly upregulated in laryngeal cancer which may serve as a biomarker for early diagnosis of laryngeal cancer.


Background
Laryngeal carcinoma is one of the most common and aggressive malignancies in head and neck region with high mortality [1,2]. Approximately 85-90% of laryngeal carcinoma cases are laryngeal squamous cell carcinoma (LSCC), which accounts for about 25% of all head and neck squamous cell carcinomas [3]. Due to the rapid industrial development and aggressive air pollution, the occurrence of laryngeal cancer exhibits increasing trend [4]. In the past three decades, the treatments for laryngeal cancer have been signi cantly improved, but the clinical outcomes of the patients have not been obviously improved [5]. One of the major reason for the unsatisfactory prognosis is delayed in diagnosis [6]. Tumor stage at initial diagnosis is a key factor for therapy decision and clinical outcomes of patients with laryngeal cancer [7]. Therefore, it is crucial to explore novel molecular biomarkers for early detection of laryngeal cancer.
Cytokines are a group of soluble, low-molecular-weight protein which play important roles in in ammatory and autoimmune responses [8,9]. In ammatory conditions can induce or promote various key processes in tumorigenesis, including oncogenic transformation, genetic alterations in malignant cells, etc [10]. Cytokines may be sensitivity biomarkers to estimate canceration in microenvironment. Interleukin-8 (IL-8), a pro-in ammatory factor of the CXC chemokine family, is located on chromosome 4q13-21, and play an important role in human cancer progression following regulating cancer invasion, angiogenesis and metastasis [11,12]. IL-8 has been reported to be involved in various human cancers, such as prostate cancer, colorectal cancer, melanoma, breast cancer, etc [13][14][15][16]. In cancer, IL-8 is mainly produced by the tumor cell themselves, thus serum concentration of IL-8 shows close association with tumor development, which may be a sensitive biomarker for early diagnosis, predicting treatment response, and evaluating prognosis in management of tumor [17]. However, the clinical signi cance of IL-8 for laryngeal cancer diagnosis is still unclear.
In present study, we mainly explored the expression level of IL-8 in laryngeal cancer, as well as its association with clinicopathological characteristics of the patients. In addition, we also investigated the diagnostic value of serum IL-8 for laryngeal cancer.

Patients
A total of 126 laryngeal cancer patients and 112 healthy controls were collected in Chinese PLA General Hospital. The laryngeal cancer patients were diagnosed by two pathologists based on histological examinations. None of the patients had received surgery, chemotherapy or radiotherapy before blood collection. The individuals in control group were healthy, without malignancy history. The case and control groups were matched in age and gender.
5 mL blood samples were collected from each participant, and then serum was separated from whole blood by centrifugation. The serum specimens were stored at -80℃ until use. The clinicopathological data were collected and listed in Table 1. Written informed consent was obtained from all patients prior to sampling. The research protocol was approved by the Ethics Committee of Chinese PLA General Hospital.

Statistical analysis
All statistical analyses were performed using SPSS 21.0 software (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). The expression level of IL-8 was shown as mean ± SD, and its comparison between patients and healthy control was performed by Student's t test. χ 2 test was used to analyze the association between IL-8 expression and the clinicopathological variables. The diagnostic value of IL-8 in laryngeal cancer was evaluated using receiver operating characteristic (ROC) curve by calculating the area under ROC curve (AUC). P value less than 0.05 was considered as statistical signi cant.

Results
Serum IL-8 expression was up-regulated in laryngeal cancer The expression levels of IL-8 in 126 patients and 112 healthy controls were tested using ELISA analysis. Serum IL-8 levels were signi cantly higher in patients with laryngeal cancer than that in the healthy controls (P < 0.05, Fig. 1).

Relationship between IL-8 and clinicopathological characteristics of laryngeal cancer
Relationship between serum IL-8 levels and clinicopathologic variables was analyzed using χ 2 test. Patients were subdivided into low (n = 56) and high (n = 70) expression groups according to their average serum IL-8 levels. Analysis results demonstrated that serum IL-8 level was positively associated with T classi cation (P = 0.031), clinical stage (P = 0.021) and lymph node metastasis (P = 0.023). However, there was no signi cant association between serum IL-8 level and other factors such as age, gender, tumor size or histological grade (all P > 0.05) ( Table 1).

The diagnostic value of IL-8 in laryngeal cancer
We used ROC curve analysis to assess the diagnostic performance of serum IL-8 for laryngeal cancer. As shown in Fig. 2

Discussion
Laryngeal cancer is a frequently diagnosed head and neck cancer. The etiology of laryngeal cancer is not very clear, but several risk factors have been con rmed to contribute progression of laryngeal cancer, including tobacco use, alcohol consumption and human papillomavirus (HPV) infection [18][19][20]. Despite the considerable progress in treatment, the mortality rate of laryngeal cancer has not been obviously improved over the previous decades, due to the low early diagnosis rate [21]. Thus, it is urgent need to identify novel biomarkers for early diagnosis of laryngeal cancer. In current study, we investigated the diagnostic signi cance of serum IL-8 for laryngeal cancer. Analysis results demonstrated that serum IL-8 could distinguish laryngeal cancer patients from healthy individuals with satisfactory sensitivity and speci city.
Laryngeal cancer is a complex process which is regulated by multiple molecular factors and signal pathways [22]. in laryngeal cancer might inhibit aggressive tumor development, thus contributing to a relatively good prognosis [24]. Kopta R et al. found that cytokines IL-23 played critical roles in determining the aggressive potential of laryngeal cancer [25]. The levels of cytokines may be sensitivity and reliable in re ecting tumor microenvironment which hold the capacity to serve as tumor biomarkers.
In this study, we investigated serum level of IL-8 in laryngeal cancer patients and healthy individuals. According to ELISA results, serum IL-8 levels were obviously higher in laryngeal cancer patients than that in healthy controls. Moreover, the elevated IL-8 level was signi cantly correlated with advanced T classi cation, high clinical stage and positive lymph node metastasis. All the data revealed that IL-8 as an oncogene might enhance the malignant progression of laryngeal cancer. The obviously increased level of circulating IL-8 in laryngeal cancer was also reported by Eyigor and colleague [26]. The study carried out by Li et al. reported that IL-8 could cooperate with stromal cell-derived factor-1 (SDF-1) and its receptor (CXCR4), thus enhancing angiogenesis and aggressive progression of laryngeal cancer [27]. However, the speci c molecular mechanisms underlying the functional roles of IL-8 in laryngeal cancer could not be explained clearly. Further studies are still required.
Given the critical roles of IL-8 in tumorigenesis, various studies have demonstrated that IL-8 had the ability to act as a biomarker for malignancy. For instance, the study carried out by Khyani et al. showed that saliva IL-8 levels were signi cantly different between healthy individuals and oral cancer patients, which might be employed as a biomarker for early detection of oral cancer [28]. In our study, ROC curve was plotted to estimate the diagnostic performance of serum IL-8 in laryngeal cancer. The high AUC, sensitivity and speci city results suggested IL-8 could discriminate between laryngeal cancer patients and healthy individuals, with high diagnostic accuracy. IL-8 might be a potential biomarker for noninvasive diagnosis of laryngeal cancer. However, due to the relatively small sample size in our study, the clinical signi cance of serum IL-8 for laryngeal cancer screening needed further investigations with large sample size.

Conclusions
In conclusion, serum IL-8 expression level is elevated in laryngeal cancer and its expression is closely correlated with tumor stage and metastasis. IL-8 may be a potential biomarker for early diagnosis of laryngeal cancer. The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study.

Consent for publication
We obtaining permission from participants to publish their data.

Availability of data and materials
All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests. approved the nal manuscript.