Salivary IL-6 levels in chronic periodontitis patients with and without oral squamous cell carcinoma: A comparative cross-sectional study at khartoum dental teaching hospital, 2016–2017

Abstract

Periodontal disease is an inflammatory disease that affects periodontal tissue, such as the gingiva, periodontal ligament and alveolar bone. Recently, a link between periodontal disease, oral cancer and inflammation has been suggested. In this study, the concentration of the inflammatory cytokine salivary IL-6 was measured to determine its role in the inflammatory process of periodontitis and oral squamous cell carcinoma. An analytical cross-sectional study was conducted at the Khartoum Dental Teaching Hospital, Sudan. Three study groups were enrolled with their consent: patients diagnosed with chronic periodontitis with at least two interproximal sites showing clinical attachment loss > 4 mm and pocket depth > 5 mm. (n = 32); patients with chronic periodontitis and oral squamous cell carcinoma (OSCC) pathology reports of OSCC at the time of saliva collection (n = 22); and matched healthy controls (n = 30). Patients were excluded if they had any chronic inflammatory condition or disease; had a history of recent trauma, acute infection, burns, laceration, previous surgery, chemotherapy, or radiotherapy; had used antibiotics within the previous three months; had a history of periodontal treatment within the previous three months; were pregnant or lactating; were taking drugs that induce hyposalivation; or had a history of previous radiation therapy to the head and neck area. A trained dentist clinically examined the participants. Periodontal parameters (plaque index, bleeding on probing, probable pocket depth and clinical attachment level) were measured, and the IL-6 concentration in the saliva samples was measured via enzyme-linked immunosorbent assay. A statistically significant difference in periodontal parameters was observed between the OSCC patient group and the other groups. OSCC patients had significantly greater IL-6 concentrations in their saliva (log 2.40 ± 0.37) pg/ml) than did chronic periodontitis patients (CP) (log 1.42 ± 0.418 pg/ml) and healthy controls (log 1.25 ± 0.13 pg/ml) (P < 0.001). High salivary IL-6 levels suggest potential utility as a biomarker for OSCC unconfounded by chronic periodontitis.

Introduction

Currently, the need to assess health status, disease susceptibility, disease progression, and treatment outcomes of patients with common medical conditions has led to the development of biomarkers (Wong 2006). According to the National Institutes of Health (NIH), a biomarker is a characteristic that is objectively measured and evaluated as an indicator of a normal biological process, pathogenic process, or pharmaceutical response to therapeutic intervention (Lesko and Atkinson 2001). These biomarkers of the host response are present in body fluids, including serum samples, saliva, and gingival crevicular fluid, and can be utilized as diagnostic markers for many oral and systemic diseases and conditions (Ebersole et al. 1997).

Cytokines are small soluble proteins produced by specific cells that cause changes in the properties or behavior of other cells locally or systemically (Okada and Murakami 1998). The cytokines include interleukins, interferons and growth factors. Cytokines are known to play a significant role in multiple biological activities, including proliferation, differentiation, development, regeneration, repair, homeostasis, and inflammation (Okada and Murakami 1998). One of the most prominent proinflammatory and procoagulant cytokines is Interleukin 6, and different cell types are responsible for its production (Bartold and Haynes 1991). The primary function of IL-6 is the initiation of the final maturation of B cells into immunoglobulin-secreting plasma cells by stimulating the secretion of antibodies to such a degree that serum IgG levels rise 120–400-fold (Yamazaki et al. 1994). Elevated levels of IL-6 in biological fluids are associated with immune pathologies, such as tissue injury, infection and inflammatory diseases (Naka et al. 2002). IL-6 may also act as an activator of transcription and signal transducer and as a growth factor for human keratinocytes and cancer cells (Page and Schroeder 1976; Barton 2005).

On the other hand, CP is an infectious disease characterized by inflammation of tooth-supporting tissues (Newman, et al. 2006). The primary etiological factor is periodontal bacterial pathogens (Newman, et al. 2006). The local host response to these bacteria includes the accumulation of leukocytes and the release of inflammatory cytokines and mediators, which play a significant role in disease pathogenesis (Prabhu et al. 1996). Inflammatory cytokines, including IL-6, IL-8 and TNF α, are strong mediators of the inflammatory process generated during periodontal pathology. The production of both IL-6 and IL-8 is controlled by nuclear factor-kappa β (NF-kβ), which is involved in inflammation, apoptosis, cell adhesion and proliferation (Mankan et al. 2009). The transcription factor NF-kβ is an early response gene that enhances the expression of a series of cytokines with proinflammatory, proangiogenic, and immunoregulatory activities and plays an essential role in carcinogenesis (Chen et al. 2001).

Head and neck cancers are the sixth most common cancer in the world(John et al. 2004), with oral squamous cell carcinoma (OSCC) representing more than 90% of all cancers(Re et al. 2010). The prevalence of oral cancer in Sudan is 3.2%, with incidence rates of 3.7% for men and 2.6% for women (Organization 2008). The diagnosis of oral squamous cell carcinoma is currently based on clinical oral examination and histological analysis of tumor tissues via an invasive sampling procedure (Jemal et al. 2008). In many cases, clinicians have difficulty detecting OSCC early, resulting in a poor prognosis and the possibility of symptomatic metastasis(Yakob et al. 2014). The role of IL-6 in both periodontitis and OSSC is well documented, and elevated IL-6 has been proposed as a biomarker for detecting OSC (Oshin, et al. 2024). IL-6 has been linked to OSCC because of its role in promoting inflammation, tumor growth, and progression. IL-6 acts by activating several signaling pathways, such as the JAK/STAT3 pathway, which contributes to tumor development by increasing cell survival, invasion, and immune evasion. Additionally, IL-6 can influence the tumor microenvironment, promoting chronic inflammation, which is a known factor in the progression of OSCC (Chagpar et al. 2016; Jaglowski, et al. 2007). Early detection of oral cancers plays a crucial role in the successful outcome of therapy. Although successful treatment and control of OSCC depend on earlier diagnosis, most diseases are not diagnosed until morbid symptoms become apparent in the late stages. Hence, the need for earlier diagnosis requires medical researchers to find molecular disease biomarkers that may help in detecting diseases before they become overt or complicated. Identifying a previously undetected disease at an early stage may ultimately save lives. Therefore, the main aim of this study was to compare the salivary levels of IL-6 in participants with oral squamous cell carcinoma with those in participants with chronic periodontitis and to investigate whether IL-6 could serve as a viable biomarker for participants with oral squamous cell carcinoma and chronic periodontitis.

Materials and methods

An analytical cross-sectional study was conducted at the Department of Periodontology and Oral Surgery at the Khartoum Dental Teaching Hospital (KDTH) in Khartoum, the capital of Sudan, over a nine-month period from December 2016 to August 2017. The study commenced following the research's approval from the ethical committees of the Sudan Medical Specialization Board (SMSB), the KTDH and the Federal Ministry of Health.

The study used total coverage of all patients who attended the clinic during the 9-month study period and were diagnosed with OSCC. Those who met the inclusion criteria were consecutively approached and included after providing consent, after which they were matched with CPs and healthy controls. A total of 84 patients were enrolled in the study, and the description of each group was as follows: Group A consisted of 22 patients diagnosed with CP and OSCC, with a mean age of 56.0 ± 13.5 years (any patient diagnosed with OSCC and fulfilling the inclusion and exclusion criteria had been invited); Group B consisted of 32 patients with chronic periodontitis, with a mean age of 42.9 ± 13.7 years; and Group C included 30 patients matched with healthy controls, with a mean age of 41.5 ± 5.5 years. The control group was selected from copatients at the same hospital and were matched for age and sex. All patients who met and fulfilled the inclusion and exclusion criteria agreed to participate and underwent full periodontal charting to rule out subclinical disease.

Data collection

Oral clinical examination

A dentist followed all infection control measures and collected medical and dental histories. Each patient was coded with a number taken to maintain patient confidentiality and data security. The periodontal parameters were obtained in a dental chair by a trained dentist via a plain dental mirror and William's graduated periodontal probe. Each patient underwent a full-mouth examination to measure the gingival index (Löe and Silness 1963), periodontal pocket depth (PPD), and clinical attachment loss (CAL) (Ramfjord 1959).

Tumor analysis

Histological examinations of biopsy-confirmed tumor types were performed by pathologists at the International Neuroendocrine Cancer Alliance (INCA). The tumor stage was determined according to the American Joint Committee on Cancer, 7th Edition (TNM)[24]. The tumor stage was established by an experienced physician who performed the first examination.

Collection of saliva samples and IL-6 measurement

Five milliliters of unstimulated whole saliva were collected from patients between 8:00 a.m. and 12:00 p.m. The participants were asked to avoid eating, drinking or performing any oral hygiene procedure, and just the participants were asked to rinse with water immediately before sample collection.

Saliva samples were immediately centrifuged at 3500 rpm for 15 min at 8 °C; the fluid phase was harvested and stored at −80 °C until the IL-6 concentration was measured. Salivary IL-6 was measured via the sandwich ELISA technique according to the supplier’s (Biolegend, USA) instructions. The ELISA for human IL-6 was performed according to the BioLegend protocol. A 215 ng/ml lyophilized IL-6 standard was reconstituted and serially diluted to produce standards ranging from 500 pg/ml to 7.8 pg/ml; 0 pg/ml served as the blank. The plates were coated overnight at 4 °C, blocked, and incubated with standards, controls, and samples. The detection antibody and avidin-HRP were sequentially added, with incubation and washing steps between. The substrate was added, and the reactions were stopped before the samples were read at 450 nm. Optical density (OD) values were averaged and used to calculate IL-6 concentrations from a four-parameter logistic standard curve constructed in Excel. The minimum detectable concentration (sensitivity) of the LEGEND MAX™ Human IL-6 ELISA Kit from BioLegend is 0.94 pg/m. The laboratory staff were blinded to all the clinical groups.

Statistical analysis

The data were analyzed via the Statistical Package for the Social Sciences (SPSS) version 21 with the assistance of a professional biostatistician.

Il-6 measurements were not normally distributed; therefore, log transformation was performed before proceeding with further analyses. One-way ANOVA was used to compare the means among the three groups, with post hoc Bonferroni correction for multiple comparisons. Additionally, when comparing the means of IL-6 between each pair of groups according to the T and N subgroup, an independent sample t test was used. Additionally, a general linear model was used to control for confounders (GI, PPD, CAL, OHI-S and snuff-dipping). The p value was set at < 0.05.

Results

Sample profile

There was age and sex matching between the groups, with a greater percentage of illiterate and married individuals. Approximately 77.3% of the cancer patients were nonsmokers, and 45.5% were nonsnuff dippers. Moreover, there was a match between the other two groups (Table 1).

Table 1.

Demographic variables of the patients in the groups under study

Demographic variables	Group A (N = 22)	Group B (N = 32)	Group C (N = 30)
	N (%)	N (%)	N (%)
Sex
Male	13 (59.1)	24 (75)	19 (63.3)
Female	9 (40.9)	8 (25)	11 (36.7)
Age in years (mean ± SD)	56 ± 13.5	42.9 ± 13.7	44.5 ± 5.5
Marital status
Single	1 (4.5)	9 (28.1)	21 (70.0)
Married	20 (91.0)	22 (68.8)	9 (30.0)
Widow(er)	1 (4.5)	0 (0)	0 (0)
Divorced	0 (0)	1 (3.1)	0 (0)
Education
Illiterate	17 (77.3)	5 (15.6)	0 (0)
Primary	2 (9.1)	6 (18.8)	0 (0)
Secondary	3 (13.6)	10 (31.3)	0 (0)
Graduate	0 (0)	11 (34.4)	30 (100)
Habits
Smokers	5 (22.7)	12 (37.5)	0 (0)
Nonsmokers	17 (77.3)	20 (62.5)	100 (100)
Snuff dippers	12 (54.5)	2 (6.3)	0 (0)
Non snuff dippers	10 (45.5)	30 (93.7)	30(100)
Oral hygiene tool
Toothbrush	18 (81.8)	31 (69.9)	30 (100)
Miswak	3 (18.2)	1 (3.1)	0 (0)

Data from Group A (22 patients with chronic periodontitis and oral squamous cell carcinoma), Group B (32 patients with chronic periodontitis), and Group C (30 healthy controls) are presented as frequencies and percentages

There was a statistically significant difference between the three studied groups for all the periodontal clinical parameters represented by the oral hygiene index-simplified (OHI-S), gingival index (GI), probable pocket depth (PPD) and clinical attachment loss (CAL) detected by the Kruskal‒Wallis H test with the Bonferroni adjustment, as shown in Table 2.

Table 2.

Comparison of periodontal parameters in the groups under study

Grouping	Group A (Mean ± SD)	Group B (Mean ± SD)	Group C (Mean ± SD)	p-value
OHIS	1.79 ± 0.34	1.51 ± 0.52	1.24 ± 0.46	0.001**
GI	1.73 ± 0.46	1.43 ± 0.4	1.34 ± 0.57	0.052
PPD	3.06 ± 0.93	1.71 ± 1.21	0 ± 0	0.001**
CAL	4.69 ± 1.59	3.2 ± 1.36	0 ± 0	0.001**

Group A (22 patients with chronic periodontitis and oral squamous cell carcinoma), Group B (32 patients with chronic periodontitis), and Group C (30 healthy controls) were used for the oral hygiene index, gingival index, probable pocket depth, and clinical attachment loss

The concentration of IL-6 in saliva

The highest concentration of IL-6, which was log 2.40 ± 0.37 pg/ml, was detected in the saliva samples of the OSCC group. The IL-6 level in the chronic periodontitis (CP) group was (log 1.42 ± 0.418) pg/ml, and that in the healthy control (HC) group was (log 1.25 ± 0.13) pg/ml. There was a strong, statistically significant difference between the periodontitis group and the periodontitis plus OSCC group (p = 0.001) as well as between the periodontitis plus OSCC group and the healthy controls (p = 0.001) (Fig. 1) and Table 3.

Fig. 1.

Levels of log-transformed IL-6 in the studied groups. P < 0.01; **P < 0.001 by ANOVA with the Bonferroni post hoc correction

Table 3.

Multiple comparisons of IL-6 levels between study groups via bonferroni correction

	Multiple Comparisons (Bonferroni corrections)
	Dependent Variable: Log- IL6
(I) group	(J) group	Std. Error	P value
Group A (OSCC Cases)	Group B (Chronic Periodontitis)	0.09111	0.000*
	Group C (Healthy control)	0.09306	0.000*
Group B (Chronic Periodontitis)	Group A (OSCC Cases)	0.09111	0.000*
	Group C (Healthy control)	0.08545	0.154
Group C (Healthy control)	Group A (OSCC Cases)	0.09306	0.000*
	Group B (Chronic Periodontitis)	0.08545	0.154

*. The mean difference is significant at the 0.05 level

Comparisons of IL-6 levels among study groups via Bonferroni correction. Group A included OSCC patients, Group B included patients with chronic periodontitis, and Group C included healthy controls. A p value less than 0.05 (marked with *) indicates a statistically significant difference after correction

A general linear model was used to compare the levels of IL-6 between the SCC group and the periodontitis group while controlling for the levels of GI, PPD, CAL, OHIS and snuff-dipping. The analysis revealed that patients in the OSCC group still had significantly higher mean IL-6 levels than patients in the periodontitis group did, with a p value of 0.000 and adjusted R squared = 0.557 (Table 4).

Table 4.

Comparison of log-transformed IL-6 levels in the oral squamous cell carcinoma group using a general linear model and controlling for GI, PPD, CAL, OHIS and scuff-dipping

Tests of Between-Subjects Effects
Dependent Variable: IL6
Source	Type III Sum of Squares	df	Mean Square	F	P value
Corrected Model	12.873a	6	2.146	12.309	0.000
Intercept	3.502	1	3.502	20.091	0.000
GI	0.006	1	0.006	0.035	0.852
PPD	0.046	1	0.046	0.265	0.609
CAL	0.017	1	0.017	0.097	0.757
Snuff-dipping	5.255E-5	1	5.255E-5	0.000	0.986
OHIS	0.005	1	0.005	0.027	0.870
Group	7.200	1	7.200	41.307	0.000**
Error	8.367	48	0.174
Total	206.619	55
Corrected Total	21.240	54

^aR Squared = 0.606 (Adjusted R Squared = 0.557)

A general linear model was used to compare the levels of IL-1 between the SCC group and the periodontitis group while controlling for the levels of GI, PPD, and CAL

In addition, the IL-6 levels were compared between those with TO & T1 (low T subgroup) and those with T2 & T3 (high T subgroup) in the OSCC group. No significant differences were detected between the 2 subgroups. Similarly, there were no significant differences in the levels of IL-6 between the N0 & N1 (low N subgroup) group and the N2 & N3 (high N subgroup) group, and the p values were less than 0.05 in both comparisons (0.852 and 0.409, respectively) (Table 5).

Table 5.

Comparison of Il-6 levels between the oral squamous cell carcinoma 2 groups according to T stage (tumor size) and N stage (lymph node involvement)

	T subgroups	N	Levene's Test sig	Mean Std. Deviation	P value
IL-6	T0&T1	3	0.202	2.28 ± 0.16	0.852
	T2&T3	19		2.46 ± 0.35
	N subgroups
IL-6	N0&N1	5	0.551	2.46 ± 0.41	0.409
	N2&N3	17		2.43 ± 0.32

The IL-6 levels were compared between those with TO & T1 (low T subgroup) and those with T2 & T3 (high T subgroup) in the SCC group, and the levels of IL-6 were compared between the N0 & N1 (low N subgroup) group and the N2 & N3 (high N subgroup) group. The p values were less than 0.05 in both comparisons (0.852 and 0.409, respectively)

Discussion

This study assessed salivary IL-6 as a potential biomarker for OSCC in patients with CP. The significantly increased IL-6 levels in OSCC patients suggest tumor-related immune dysregulation, although periodontitis may still influence biomarker reliability.

The mean age of the participants in the present study was in the middle 40 s, which is similar to the age groups reported by Juretic et al. (2013) (Jureti et al. 2013). Chronic periodontitis starts developing around the age of 35 years (Wiebe and Putnins 2000). The mean age of the cancer patients was approximately the fifth decade. This finding is also reported in the literature, which indicates that cancer is a disease of elderly individuals, and most epidemiological studies have demonstrated that oral squamous cell carcinoma occurs between the fifth and seventh decades of life (Fan et al. 2014).

The results of this study demonstrated that salivary IL-6 levels are significantly higher in OSCC + CP patients than in CP patients or controls, suggesting the possibility of using IL-6 as a biomarker for oral squamous cell carcinoma; prospective validation is needed. This result is similar to the results of several studies that support this hypothesis (Arellano-Garcia et al. 2008; Lisa Cheng et al. 2014; Sato et al. 2010; Riedel et al. 2005). St John et al. (2004) demonstrated no significant differences in salivary IL-6 between oral cancer patients and healthy controls. They reported that IL-8 was detected at relatively high concentrations in the saliva and that IL-6 was detected at relatively high concentrations in the serum of patients with oral squamous cell carcinoma. However, they did not consider the gingival or periodontal condition, so they proposed that the contribution of OSCC to the elevation of IL-8 and IL-6 in saliva exceeded any background contribution from the host’s inflammatory conditions (John et al. 2004).

The role of IL-6 in the inflammatory response in periodontitis was evident by the significant increase in its concentration in saliva samples from the CP group compared with samples from healthy controls (59.29 ± 128.34 and 18.85 ± 6.26 pgm/ml, respectively). Similar findings were reported by Costa (2010) and Liskmann (2006), who reported increased salivary levels of IL-6 in patients with CP compared with healthy controls (Costa et al. 2010; Liskmann, et al. 2006; Ebersole and Cappelli 2000). However, Giannopoulou and Wang (2003) reported that salivary and serum levels of the proinflammatory cytokine IL-6 may be increased as a result of various oral cavity inflammatory conditions, not just periodontitis (Wang et al. 2003). Additionally, other studies of salivary IL-6 demonstrated no association with periodontitis (Rathnayake et al. 2013).

Periodontitis is a serious oral pathology affecting periodontal tissues accompanied by a local inflammatory response similar to the response reported in patients with OSCC. The similarity of the inflammatory response in periodontitis and oral cancer pathology suggests that common host immune factors are involved in the pathogenesis of these two diseases (Lax and Thomas 2002; Karin et al. 2006).

In our study, there was no difference in the concentration of interleukin 6 in different stages of cancer (T1, T2 and T3, T4) because of the limited sample size of the 2 subgroups. Furthermore, there was no significant difference in any of the periodontal parameters between the two groups. This result may support the hypothesis that IL-6 affects the progression of cancer, which is in agreement with a study that proposed that the majority of IL-6 in saliva comes from local tissues, so large tumors secrete greater amounts of IL-6 (Sun et al. 2002).

Similarly, the studies of Brailo et al. and Nishimoto et al. reported no significant correlation between IL-6 levels and T stage. They reported that IL-6 is produced by various cell types, including lymphoid cells, monocytes, fibroblasts, endothelial cells, oral epithelial cells, and several types of tumor cells (Brailo et al. 2006; Nishimoto et al. 2000).

With respect to the degree of invasion of regional lymph nodes (N-staging), no significant findings were reported, in contrast to some studies that reported an association between IL6 expression and the rate of lymph node metastasis in OSCC patients (Chen et al. 2012; Shinriki et al. 2011).

In summary, given that saliva is in direct contact with the oral mucosa and cancerous lesions in the oral cavity, salivary diagnostics for periodontal disease are promising and could facilitate early diagnostic and treatment in clinical practice. Additionally, the screening and detection of early OSCC lesions via saliva look promising. Salivary diagnostics may avoid many unnecessary biopsies and hospital and outpatient clinic visits. Existing therapies for OSCC patients are based on traditional stage-predicting guides (mostly the TNM criteria) and histological grading; various diagnostic tests are commercially available, and the development of point-of-care tests is expanding and could be useful adjuncts or substitutes for these methods.

In our study, there are several limitations. First, the sample size was selected according to previous literature; for future studies, it is better to calculate the desired sample size according to the powered design in the study, although we did so as much as possible to recruit patients with OSCC with strict exclusion criteria. Additionally, in patients with OSCC, CP was diagnosed just before surgery, with no earlier records of chronic periodontitis. Furthermore, IL-6 is a broad inflammatory marker that is elevated in infections, ulcers, and systemic inflammation; thus, with this limited specificity, we suggest combining IL-6 with other biomarkers (IL-8 and MMP-9).

Conclusion

1. Salivary IL-6 was significantly greater in the OSCC group than in the CP and control groups.

2. IL-6 remained high in the OSCC group even after the use of the general linear model in the control of confounders (GI, PPD, and CAL)

3. There was no significant difference in the OSCC group according to the stage of cancer (T) or lymph node involvement (N).

Recommendations

1. Further studies with larger sample sizes and stronger evidence are needed to confirm the utility of these cytokines in the diagnosis of OSCC.

2. More research efforts are necessary to determine the sensitivity and specificity of salivary biomarker detection.

3. Early prevention and treatment of CP are essential for maintaining oral health and preventing potentially lethal diseases such as OSCC.

Authors contributions

The study was conceptualized and designed by A. A. A., A. M. R, M. M. M, M. O. A. A, and D. A. A. A. A.A. was responsible for participant recruitment from Khartoum Dental Teaching Hospital and the coordination of data collection. All the authors managed the project logistics, including obtaining ethical approval and preparing the questionnaire materials, and contributed to the discussion section of the manuscript. A. A. A. participated in the interpretation and discussion of findings and conducted the data analysis. The manuscript was initially drafted by A. A. A. and was subsequently reviewed, edited, and revised by all coauthors. All the authors have read and approved the final version of the manuscript.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request. The data were anonymized to ensure participant confidentiality.

Declarations

Ethics approval and consent to participate

The study protocol was reviewed and approved by the Ethics Committee of the Sudan Medical Specialization Board (SMSB), the KTDH and the Federal Ministry of Health. Written informed consent was obtained from all participants prior to sample collection. Participation was voluntary, and confidentiality was strictly maintained throughout the study.

Consent for publication

All participants signed a consent form indicating their agreement to participate in the study and to allow the anonymous use of their data for publication and research purposes.

Competing interests

The authors declare that there are no competing interests related to this study.