|HEAD AND NECK SYMPOSIUM - ORIGINAL ARTICLE
|Year : 2014 | Volume
| Issue : 3 | Page : 227-230
Serum levels of interleukin-7 and interleukin-8 in head and neck squamous cell carcinoma
Z Mojtahedi1, B Khademi2, N Erfani1, Y Taregh1, Z Rafati1, M Malekzadeh1, A Ghaderi1
1 Department of Immunology, Institute for Cancer Research, Shiraz University of Medical Sciences, School of Medicine, Shiraz, Iran
2 Department of Otorhinolaryngology, Khalili Hospital, Shiraz University of Medical Sciences, Shiraz, Iran
|Date of Web Publication||10-Dec-2014|
Department of Immunology, Institute for Cancer Research, Shiraz University of Medical Sciences, School of Medicine, Shiraz
Source of Support: None, Conflict of Interest: None
Background: The aim of this study was to investigate the association of head and neck squamous cell carcinoma (HNSCC) with serum levels of interleukin-7 (IL-7) and IL-8, the two cytokines whose associations with HNSCC need more clarifications. Materials and Methods: Commercial enzyme-linked immunosorbent assay kits were used for the quantification of the cytokines. Sera were collected from 48 untreated patients (36 men and 12 women; mean age: 52.7 ± 9.8 years) and 34 healthy donors (26 men and 8 women; mean age: 53.1 ± 9.0 years). Results: Serum IL-8 level was neither significantly different between HNSCC patients and control individuals nor associated with smoking status, gender, age, tumor location, tumor grade, and stage of the patients (P > 0.05). Regarding IL-7, all control individuals had serum levels below the sensitivity of the kit (3 pg/ml), but nine patients had detectable levels, and that the mean serum IL-7 was significantly higher in the patients compared to the controls (P = 0.008). Conclusions: Serum IL-8 level is not significantly associated with HNSCC. With the sensitivity of the kit we employed, it seems that serum IL-7 levels are specifically elevated in HNSCC patients compared to healthy individuals. Data from other independent studies are required to clarify the possible employment of IL-7 as an HNSCC biomarker.
Keywords: Enzyme-linked immunosorbent assay, head and neck neoplasms, interleukin-7, interleukin-8, serum
|How to cite this article:|
Mojtahedi Z, Khademi B, Erfani N, Taregh Y, Rafati Z, Malekzadeh M, Ghaderi A. Serum levels of interleukin-7 and interleukin-8 in head and neck squamous cell carcinoma. Indian J Cancer 2014;51:227-30
|How to cite this URL:|
Mojtahedi Z, Khademi B, Erfani N, Taregh Y, Rafati Z, Malekzadeh M, Ghaderi A. Serum levels of interleukin-7 and interleukin-8 in head and neck squamous cell carcinoma. Indian J Cancer [serial online] 2014 [cited 2019 Nov 15];51:227-30. Available from: http://www.indianjcancer.com/text.asp?2014/51/3/227/146728
| » Introduction|| |
Head and neck squamous cell carcinoma (HNSCC) originates from the mucosal surfaces of the upper aerodigestive tract including the oral cavity, pharynx, and larynx. The disease mortality rate is still high that is partly due to the late diagnosis and inefficient therapy.  For reducing the mortality, it is critical to identify novel HNSCC biomarkers with potential utility in diagnosis and/or therapy guidelines.
Serum has been recognized as the most ideal source for biomarkers because of its trouble-free access and high amount.  Serum cytokine levels have been associated with initiation, progression, prognosis, and response to therapy in malignant processes. , Interleukin-7 (IL-7) and IL-8 are two cytokines whose associations with HNSCC need more clarifications. In contrast to a small number of the literature published regarding IL-7 and HNSCC, several publications have discussed serum IL-8 and HNSCC, but the results are conflicting. ,,,,,,,
Interleukin-7 is mainly produced by stromal and parenchymal cells, and to a lesser extent by immune cells such as dendritic cells. This pleiotropic cytokine is a crucial factor for development and differentiation of T cells from its progenitors, survival of naïve T cells, and expansion of CD4+ and CD8+ T cells.  Contrary to these immune-stimulating properties that might be in favor of tumor protection, IL-7 can promote tumorigenesis. Al-Rawi et al.  showed the growth factor activity of IL-7 for both endothelial and breast cancer cells. This activity is thought to be mediated through a paracrine manner because cancer cells express the IL-7 receptor, but no significant amount of IL-7. , The tissue overexpression and increased serum IL-7 levels have also been reported in a variety of cancers. ,, For example, in breast cancer, the tissue overexpression of IL-7 was found to be a poor prognostic biomarker.  In addition to a prognostic value, in ovarian cancer, increased serum levels of IL-7 showed a diagnostic value.  A few reports have been published in the case of IL-7 in HNSCC. , Linkov et al. tested concentrations of 60 cytokines, growth factors, and tumor antigens in the sera of HNSCC patients and compared them to those in a healthy group. IL-7 was among the panel of the 25 multi-markers offering the highest diagnostic power for HNSCC. 
IL-8 CXCL8 is a pro-inflammatory CXC chemokine, which promotes neutrophils chemotaxis and degranulation. Overexpression of IL-8 and/or its receptor have been reported in endothelial cells, infiltrating neutrophils, and tumor-associated macrophages.  Both IL-8 and its receptor are expressed by several cancer cell lines including HNSCC cells. IL-8 through its receptor on tumor cells, autocrine pathway, can promote tumor migration and invasion, and through its receptors on endothelial cells, paracrine pathway, contributes to angiogenesis. , Despite evidence shows an important role for IL-8 in the tumor microenvironment in HNSCC , the usefulness of serum IL-8 levels as an HNSCC biomarker is the subject of controversy. Although some reported the association of serum IL-8 with HNSCC, others did not find such associations. ,,,,,
Pre-treatment serum IL-4, IL-6, IL-10, and IL-18 levels in Iranian HNSCC patients have been previously studied. , Consistent with other studies, it was found that serum IL-6 level is associated with HNSCC development and progression.  As the association of serum IL-7 and IL-8 levels with HNSCC needs more clarification, we aimed to compare the pre-treatment serum levels of IL-7 and IL-8 in Iranian HNSCC patients to those in healthy control individuals. Also the possible association of serum levels of these two cytokines with gender, age, smoking status, tumor location, tumor grade, and stage at diagnosis in HNSCC was evaluated.
| » Materials and Methods|| |
The study was approved by the local Ethics Committee. All participants were informed that blood samples would be used in research projects, and their consent was obtained.
Patient group comprised of 48 non-related, newly diagnosed HNSCC cases (36 men and 12 women; mean age: 52.7 ± 9.8 years and age range from 36 to 74). Squamous cell carcinoma was verified histopathologically. Their characteristics including, gender, age, smoking status, tumor location, grade, and stage at diagnosis were obtained from their files. The stage of the disease was determined according to tumor-node-metastasis TNM classification. 
The control group was 34 non-related healthy individuals (26 men and 8 women; mean age: 53.1 ± 9.0 years and age range: 35-73). Control individuals were members of our university or blood donors who came to local Blood Transfusion Center. They were apparently healthy with no history of malignant, metabolic, or autoimmune diseases.
The number of patients and controls with a smoking history of less than 10 years was too low. As we wanted a more homogenous group regarding smoking status, they were not included in our study. Every participant was interviewed and examined regarding symptoms and signs of an infection, such as cough, fever, and throat inflammation. Routine laboratory tests such as cell blood count CBC were also done for patients and blood donors of the Transfusion Center. No evidence of an acute infection in the past 1 month was found in any of the participants.
Serum interleukin-7 and interleukin-8 assays
After taking blood from 48 newly diagnosed patients and 34 healthy volunteers, the sera were collected in maximum of 2 h from sampling. The collected sera were aliquoted and stored at -70°C until use. The storage time for all sera was less than 2 years.
The serum levels of IL-7 and IL-8 were determined in duplicate using commercial enzyme-linked immunosorbent assay (ELISA) kits (eBioscience, USA) according to the manufacturer's protocols. The minimum detectable limits of the kits were 3 pg/ml and 1.3 pg/ml for IL-7 and IL-8, respectively. Concentrations below the detection limits were considered zero in statistical analyses in both patient and control groups.
The data were analyzed using Statistical Package for the Social Sciences (SPSS) software (version 11.5.0; SPSS, Chicago, IL, USA). Levene's test for equality of variances indicated an equal variance for IL-8 levels but not for IL-7. Therefore, parametric t-test or analysis of variance (ANOVA) tests, where appropriate, were used for analyses of IL-8 levels, and Mann-Whitney U-test for analyses of IL-7 levels.
As the percentages of our smoker patients were higher than our control group (68.8% vs. 44.1%), the effect of cigarette smoking (smoking vs. non-smoking) on IL-8 in patient and control groups as a possible confounding factor was tested using univariate analysis, in which, IL-8 level was set as the dependent variable, and smoking status and our groups (patients and controls) as fixed factors. The model was set to main effects. Findings were considered statistically significant at a P < 0.05.
| » Results|| |
Of the 34 controls, 15 were smokers and others were non-smokers. The history of smoking status was available for 32 out of 48 patients, of which, 22 were smokers and 10 were non-smokers. Our smoker group was current smokers who smoked more than 10 years, and the never smoker group had no history of smoking.
The serum levels of IL-7 and IL-8 were measured using ELISA commercial kits. Nine patients had detectable levels of IL-7 (>3 pg/ml), whereas IL-7 was not detectable in the healthy group. Serum IL-8 levels were detectable (>1.3 pg/ml) in 38 patients and all healthy individuals. [Table 1] indicates median and mean serum levels of IL-7 and IL-8 in patients and controls. The serum IL-7 levels in HNSCC patients were significantly higher than those in healthy controls (P = 0.008). However, serum IL-8 levels did not significantly differ from those in healthy group. Univariate analysis revealed no significant effect of smoking status on IL-8 levels in patients and control groups (P = 0.2). The means and standard deviations of IL-8 in smoker and non-smoker HNSCC patients were 35.0 ± 99.2 and 18.6 ± 23.7, respectively. These values in control group were 61.1 ± 53.4 in smokers and 35.1 ± 34.2 in non-smokers.
|Table 1: Serum levels of interleukin-7 and interleukin-8 in head and neck squamous cell carcinoma patients and healthy controls|
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Due to the low numbers of patients with detectable IL-7 levels, no statistical analysis was made in terms of the association of IL-7 with clinicopathological characteristics of the disease. The descriptions of the patients are shown in [Table 2].
|Table 2: Descriptions of 9 out of 48 head and neck squamous cell carcinoma patients whose serum interleukin-7 levels were detectable|
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The means serum levels of IL-8 in the patient group according to gender, age, smoking status, grade of the tumor, tumor location, and stage are indicated in [Table 3]. IL-8 levels were not significantly associated with these parameters.
|Table 3: Serum interleukin-8 levels according to characteristics of the 48 head and neck squamous cell carcinoma patients|
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| » Discussion|| |
Serum cytokine levels have drawn attention as diagnostics, prognostics, and therapy guidelines in cancer. , Here, we tested the associations of serum levels of IL-7 and IL-8 with HNSCC. A few reports have been published on the topic of the serum IL-7 and HNSCC; nevertheless, there are several reports on serum IL-8 and HNSCC, but the results are conflicting. ,,,,,,,
In our study, IL-7 was detected in sera from 19% of the patients but in no control healthy individuals, where the difference was statistically significant. In agreement with our results, Linkov et al. investigated serum levels of 60 potential biomarkers to identify the profile of HNSCC biomarkers, and found serum IL-7 level among 25 multi-markers discriminating patients with HNSCC from healthy smokers. However, they have not studied the associations of IL-7 with clinicopathological features of the patients.  In tissue, using immunohistochemistry of snap-frozen tumor specimens from 25 patients with HNSCC, Paleri et al. showed the expression of IL-7 in all tumor samples, and the expression was not related to tumor stage or site. High expression of IL-7 showed a tendency toward a better survival.  Because of low numbers of our patients with detectable levels of IL-7, we did not statistically analyze the associations of IL-7 with clinicopathological features of HNSCC.
In contrast to IL-7 and HNSCC, there are more publications discussing the associations of IL-7 with some other types of cancer such as prostate, breast, and ovarian cancers. ,, For example in ovarian cancer, serum IL-7 levels were higher in the patients compared to both healthy control group and benign tumor patients, and also was related to more advanced disease and residual tumor after surgery. A cutoff point of 3.8 pg/ml gave the highest accuracy to distinguish malignant from benign ovarian tumors.  In our study, the detection limit of the kit was 3 pg/ml. We observed 100% specificity and 19% sensitivity for IL-7. Whether kits with a lower detection limit provide more sensitivity for serum IL-7 in HNSCC needs more investigations.
Here, serum IL-8 levels did not show statistically significant different between patients and healthy control group. Moreover, serum IL-8 levels were not associated with smoking status, gender, age, tumor location, grade, and stage. Similarly, in the study conducted by Hong et al., serum IL-8 levels in patients did not differ from those in controls (88.79 ± 223.36 in patients vs. 154.46 ± 195.02 in controls), and were not associated with gender, age, tumor grade, stage, or location too.  St John et al. also studied 19 patients with newly diagnosed T1 or T2 oral cavity or oropharyngeal carcinoma and 32 age- and sex-matched disease-free subjects with comparable smoking histories as controls. The levels of IL-8 in serum did not differ between patients and controls. They did not detect differences in IL-8 levels between smoking and non-smoking subjects, as well.  Others reported a significant association of IL-8 levels with HNSSC development or its clinicopathological manifestations. Druzgal et al. found that the difference in the mean serum IL-8 between normal controls and patients was not statistically significant. They also found no association between smoking or tumor stage and serum IL-8 levels. However, serum IL-8 levels were observed to be consistently elevated in patients with recurrent or metastatic disease  the parameters that were not assessed in our study. Linkov et al., Dung et al., Hoffmann et al., and Melinceanu et al. showed IL-8 levels were significantly higher in patients than in healthy controls. ,,, The two former studies presented no data about the stage or other characteristics of the patient group. , In the third study, no statistically significant association was found between T status, N status, and time to recurrence and serum IL-8 levels.  The latter study found that heavy smoking was significantly associated with higher levels of IL-8. 
The above observations indicate that serum IL-8 level is possibly influenced by the individual heterogeneity of HNSCC. However, IL-8 might be still a soluble, easy detectable biomarker candidate in HNSCC in other body fluids. For example, St John et al.  reported higher levels of IL-8 in saliva, but not in serum, from patients with HNSCC compared to age- and sex-matched healthy controls. Serum IL-8 might be a better candidate for other types of cancer such as lung with which a strong association was found. 
The concept of individualized therapy is emerging for the optimal therapy of cancer patients.  The highest level of IL-8 in our studied group was observed in a laryngeal carcinoma smoker, who was the only patient with the levels above the upper limit of the control group. This may indicate that serum IL-8 levels might be important in the pathogenesis of the disease in a subset of patients. The possible employment of serum IL-8 as a therapy guideline for individualized therapy needs more investigations.
Taken together, analysis of serum levels of IL-7 and IL-8 in Iranian HNSCC patients revealed that IL-7 levels, but not IL-8, were significantly elevated in HNSCC patients. Current knowledge about serum IL-7 level and HNSCC is limited to a few publications. Our results warrant further investigation of serum IL-7 and HNSCC in different populations to clarify its potential as an HNSCC biomarker.
| » Acknowledgments|| |
This study was supported by the Shiraz University of Medical Sciences (88-01-01-1902 and 88-01-01-1877) and by the Institute for Cancer Research (ICR-82-93). The study was partly based on two MD theses (Yekta Taregh and Zahra Rafati) submitted to the Shiraz University of Medical Sciences.
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[Table 1], [Table 2], [Table 3]
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