Correlation Between Periodontal Disease and Oral, Oropharyngeal, and Parapharyngeal Cancers

Introduction

Periodontal disease is a persistent inflammatory condition that impacts the bone and tissues that support the tooth. It leads to the deterioration of tissues that support the tooth, heightened tooth mobility, and, in some cases, tooth loss.^1–3 The occurrence of gingival tissue damage may be attributed to a disparity between a regional infection and excessive inflammation from the host.^4–6 The previous researches suggest that the impact of periodontal disease may extend beyond the mouth and have broader consequences throughout the body.^7–9 Oral cancer is a complex inflammatory illness that arises from a combination of several risk factors, including genetics, socioeconomic factors, and lifestyle choices.¹⁰ Cancers of the head and neck region are characterised by a local inflammatory reaction.¹¹ This response can trigger changes in cellular processes that facilitate the invasion and growth of cancer cells in primary oral carcinomas. Additionally, it can facilitate the spread of tumour cells to the gingival tissue, leading to the formation of secondary tumours.¹² Interleukin-8 is a neutrophil-activating chemokine that is essential for neutrophil extravasation from gingival capillaries and movement to the periodontal sulcus.¹³ Excessive interleukin 8 (IL-8) production results in improper spreading and emigration of neutrophils to affected areas, which is linked to substantial periodontal tissue degradation.¹⁴ It has been shown that this chemokine exhibits pro-tumoral effects linked to establishing and progressing the malignancies.^15–18

Tumour cells may express several cytokines and associated receptors that promote inflammation and induce cancer.^19,20 Some studies showed that IL-8 expression in patients with breast cancer, malignant ovarian and osteo-carcinoma is associated with metastasis of cancer cells,^21,22 advanced diseases, poor survival, and early recurrence.^23–25 Nuclear factor kappa B (NF-κB) is an intracellular transcription factor that has a unique role in a number of inflammatory and cancer-related processes.²⁶ Extrinsic stimuli, including cytokines, growth factors, oncoproteins, and stress signals, may trigger NF-κB pathways.²⁷ Few investigations assessed the expression of NF-κB in the colonic tissue of patients with colonic polyps and colorectal cancer.²⁸ Moreover, periodontal pathogens are correlated with the development of cancer in areas other than the gums, particularly the side edge of the tongue, which is the most common location.²⁹ Previous studies establish the etio-pathological connections between chronic periodontitis and oral carcinoma or oropharyngeal cancer.^30,31 The chemotactic cytokines have a role in host-microbiome interactions in periodontal disease and oral cancer.³² To date, no previous study has evaluated the correlation between oral, oropharyngeal, and parapharyngeal cancers with periodontitis (CA-with-P); oral, oropharyngeal, and parapharyngeal cancers without periodontitis (CA-without-P); and periodontitis (P) patients in salivary IL-8 and NF-κB levels and scores of clinical periodontal parameters detecting inflammation and periodontal pathogenies accumulation. This aspect motivated the present study, whose aim was to evaluate clinical periodontal parameters (PLI, BOP, PPD, CAL) and determine IL-8 and NF-κB levels from saliva, followed by an assessment of the correlation between periodontitis severity and oral, oropharyngeal, and parapharyngeal cancers.

Materials and Methods

This case study ranges between 2021 to 2024. The study comprised 300 participants from the Al-Hussein Medical City and Al-Hassan Al-Mujtabaa Hospital in Karbala, Iraq. The assessment was conducted on a consecutive series of 150 patients with cancer lesions divided into two groups: cancers with periodontitis (CA-with-P) and cancers without periodontitis (CA-without-P). The periodontitis (P) and control (C) groups comprised 75 individuals each. The ages of the male and female participants ranged between 40–65 years. The primary lesions of the oral cavity, oropharynx, or parapharyngeal spaces were resected surgically.

The criteria for inclusion and exclusion included: patients had to have at least 20 teeth; had not undergone any periodontal therapy in the preceding six months; had no lip malignancies, human immunodeficiency virus (HIV), or chronic inflammatory illnesses, such as rheumatoid arthritis or Crohn’s disease; had not taken any anti-inflammatory medications in the preceding three months; did not have diabetes, smoking habits, alcohol intake, or a family history of oral and oropharyngeal cancer, mento-labial region or pre-existing anomalies, grossed offensive of oral malignancies, or previously received radiation treatment for oral or oropharyngeal cancers.

Periodontal Examination

A single, qualified, and knowledgeable examiner conducted the periodontal examinations. For probing pocket depth (PPD) and clinical attachment loss (CAL), the intra-examiner reliability calibration was 95%. In line with the 2017 classification scheme to assess periodontitis severity, the participants were examined to ensure that the P and CA-with-P groups had an interdental CAL of ≥2 non-adjacent teeth or CAL of ≥3 mm on the buccal (facial) or lingual/palatal aspects, a PPD of >3 mm at more than two teeth, had stages III and IV periodontitis, and had grades B or C with unstable cases, meaning a PPD of ≥4 mm with bleeding on probing (BOP) or PPD of >5 mm with or without BOP.

Probing pocket depth (PPD), CAL, BOP, and plaque index (PI) were among the periodontal indicators. All teeth were assessed for clinical characteristics, with the exception of third molars. Probing pocket depth (PPD) and CAL were assessed at six places per tooth, whereas PI and BOP were recorded at four sites. The plaque index (PI) was regarded as either present or nonexistent.³³ The presence or lack of bleeding following probing was used to identify BOP.³⁴ A standardised periodontal probe (UNC-15) was used throughout the evaluations. At least two locations with CAL ≥6 mm and PPD ≥7 mm at the proximal sites of two distinct teeth were considered indicators of severe periodontitis.³⁵ The C group had clinically healthy periodontium, BOP <10%, PPD ≤3 mm, and intact periodontiums.³⁶

Tumour Examination

All patients were evaluated preoperatively through clinical examination and a radiographical assessment that included panoramic radiography and computed tomography examinations. The latter provided information about the location of the malignant sites in relation to the landmark of anatomy. Histopathological examination of the biopsy specimens was carried out by medical pathologists who specialise in evaluating tumours. The precise position of each tumour was determined using the third version of the International Classification of Diseases for Oncology.³⁷ The present study was designed to include only medically fit patients who had oral, oropharyngeal or parapharyngeal lesions that required surgical excision prior to chemotherapy or radiotherapy. The patients were categorised into three groups based on the region requiring treatment, as demonstrated in Table 1.

Table 1 Participant Division Based on Cancer Lesion Location

Saliva Sample Collection and Analysis

The participants were encouraged to brush their teeth and floss before their saliva was collected. Unstimulated saliva samples were collected between 09:00 and 12:00. Passive drooling method of saliva collection. The patients were asked to swallow saliva first, and then allow the saliva to drain passively for five minutes over the lower lip into a clean plastic cup. A micropipette was then used to transfer 300μL of the saliva samples into an Eppendorf tube (Thermo Fisher Scientific), which was then centrifuged at 3000×g for 20 mins and stored at −20°C. The protein concentration of each biomarker was measured using an enzyme-linked immunosorbent assay (ELISA). Commercially available My BioSource ELISA kits were used to determine the levels of IL-8 and NF-κB in the saliva by following the instructions specified by the manufacturer. Antigen can be detected by sandwich ELISA. In this technique, an antibody is coated on the microtiter well. A sample containing antigen is added to the well and allowed to react with the antibody attached to the well, forming an antigen-antibody complex. After the well is washed, a second enzyme-linked antibody specific for a different epitope on the antigen is added and allowed to react with the bound antigen. Then after unbound secondary antibody is removed by washing. Finally, a substrate is added to the plate which is hydrolyzed by an enzyme to form colored products. The absorbance of all proteins was measured using a spectrophotometer plate reader. The detection limits for IL-8 and NF-κB were 2.05pg/mL and 0.15ng/mL, respectively.

Statistical Analysis

IBM’s SPSS Statistics version 22 was used to perform chi-square and analysis of variance (ANOVA) tests for statistical evaluation. Chi-square is a statistical test that can be used to investigate the correlation between two qualitative variables. In the case of scaled variables, ANOVA tests were utilised to discover significant variations. Independent T-test for intragroup comparison. Logistic regression was used to analyse the relationship between periodontitis and malignancies affecting the oral, oropharyngeal, and parapharyngeal regions in the various variables of IL-8, NF-κB and clinical periodontal parameters. Statistical significance was set at a probability ≤ 0.05.

Results

The data of the 300 subjects enrolled in the present study were analysed. It was revealed that the mean age and gender did not significantly differ across the groups. The mean PI, BOP, PPD, and CAL of the CA-with-P and P groups were considerably higher than the C and CA-without-P groups (Table 2). The CA-with-P, CA-without-P, and P groups had higher levels of salivary IL-8 and NF-κB than the C group, as shown in Figures 1 and 2. The CA-with-P group had a higher incidence of oral cavity (A) and oropharyngeal (B) cancers than the CA-without-P group. The CA-with-P group had slightly lower incidences of parapharyngeal (C) cancers than the CA-without-P group (Table 2), as illustrated in Figure 3.

Table 2 The Demographic Characteristics and Clinical Periodontal Parameters of the Participants

Figure 1 Salivary Mean of IL-8 (pg/mL) Levels in Patient Groups (Periodontitis, CA-without-P: Cancer without periodontists, CA-with-P: Cancer with Periodontitis) and Control Group.

Figure 2 Salivary Mean of NF-kB Levels (ng/mL) in Patient Groups (Periodontitis, CA-without-P: Cancer without periodontists, CA-with-P: Cancer with Periodontitis), and Control Group.

Figure 3 The different types of cancers according to the location distributed in patient groups (Periodontitis, CA-without-P: Cancer without periodontists, and CA-with-P: Cancer with Periodontitis). Oral cavity (A), Oropharyngeal (B), and Parapharyngeal Region (C).

The Present Study Evaluated 150 Cancer Cases Based on Different Tissue Types and Origins (Table 3). Intragroup comparisons between subgroups of oral cavity (A), oropharyngeal (B) and parapharyngeal (C) cancers across P, CA-without-P group, and CA-with-P group are presented in (Table 4). There were significant differences (p< 0.05) between subgroups of oral cavity (A) and parapharyngeal (C) cancers and between oropharyngeal (B) and parapharyngeal (C) cancers in the CA-without-P group. Additionally, there were significant differences (p< 0.05) between subgroups of the oral cavity (A) and oropharyngeal (B) cancers in the CA-with-P group. While there were non-significant differences among sub-groups of the oral cavity (A), oropharyngeal (B) and parapharyngeal (C) cancers in the P group; between oral cavity (A) and oropharyngeal (B) cancers in the CA-without-P group. As well as between oral cavity (A) and parapharyngeal (C) cancers and between oropharyngeal (B) and parapharyngeal (C) cancers in the CA-without-P group.

Table 3 The Cancer Lesion of the Participants According to the Different Types of Tissues of Origin

Table 4 Intragroup Comparisons Between Subgroups of Different Cancer Lesion Locations in the Periodontitis Group, Cancer with Periodontitis Group and Cancer with Periodontitis Group

The correlation between periodontitis and cancers was studied using multiple logistic regression. After logistic regression models have been adjusted for age or gender. The result revealed a significant correlation between clinical periodontal parameters, lesion locations, and IL-8 and NF-κB levels with periodontitis severity and cancers (Table 5).

Table 5 The Multiple Logistic Regression Model-Based Correlations Between Periodontitis Severity and Cancers

In logistic regression analyses, (a) represented all periodontitis and cancer cases to signify the severity of both diseases, (b) represented the controls assigned as control status and the CA-with-P and CA-without-P groups as case state in logistic regression analyses to indicate cancer lesions, and (c) represented periodontitis cases as control state in logistic regression analyses and the case state included the CA-with-P and CA-without-P groups to recognise the association between both diseases. This means the connection between periodontitis and specific cancer types is a significant association. Thus, the localized inflammation in periodontal disease with elevated periodontal parameters of inflammation (BOP, PPD, CAL), increased periodontal-pathogen accumulations (PI), and increased IL-8, NF-κB levels drive the development, propagation, and advancement of tumour formation.

Discussion

Periodontal disease and oral malignancies are two of the most prevalent inflammatory illnesses. Periodontal disease arises from a bacterial infection, and the bacteria contributes to the onset of cancers in the oral regions.³⁸

The present study is the first to assess the mechanisms underlying the correlation between these two disorders, as elucidated by higher levels of salivary IL-8 and NF-κB. They positively and significantly correlate with the severity of clinical periodontal parameters. The results of the present study indicate higher mean values of clinical periodontal parameters in the CA-with-P and P groups than in the CA-without-P and C groups.

Principally, the current study’s findings indicate a considerable rise in periodontal parameter scores of PI and BOP in the P and CA-with-P groups. Periodontal bacteria, which contributes to the development of periodontal disorders, could have a major impact on the onset of oral cancers. First, the evidence indicates the involvement of bacterial infection in the development, propagation, and advancement of cancer.³⁹ Furthermore, oral cancers may be significantly influenced by periodontal bacteria, which causes periodontal disorders.⁴⁰ The majority of bacteria in plaque are part of the natural flora. Nonetheless, several bacteria found in the plaque of unhealthy periodontium have been recognised as potential pathogens. Oral neoplasms frequently harbour periodontal pathogens.⁴¹ Numerous studies have shown that people with periodontal disease have a two- to five-fold higher chance of developing oral cancers than those without the disease.^42,43 Probing pocket depth (PPD) and CAL were also higher in the P and CA-with-P groups.

This lends credence to Sahingur et al’s hypothesis that oral squamous cell carcinoma (OSCC) develops in an environment conducive to periodontal disease severity and alterations in the oral microbiome.⁴⁴ The present study indicates that periodontitis severity and malignancies of the mouth, throat, and parapharyngeal significantly correlate. The findings of the present study are also in agreement with Moraes et al’s findings that there was a substantial correlation between periodontitis and the incidence of oral cancers, even after adjusting for typical confounding variables, such as smoking, alcohol intake, and human papillomavirus.⁴⁵ Another study discovered that periodontal disease-related tooth loss due to decreased bone mass is an additional risk variable for head and neck cancer.⁴⁶ Moreover, individuals with periodontitis showed elevated levels of telomerase reverse transcriptase, an indicator of extremely selective expression of malignant cells.⁴⁷ The immune-inflammatory responses play essential roles in the patient’s susceptibility to periodontal diseases and oral cancers.⁴⁸ Therefore, it can be suggested that periodontitis severity may act as a hazard factor for oral, oropharyngeal, and parapharyngeal cancers. The present study indicates that there may be a reciprocal or bilateral correlation between periodontal disease and oral cancers. Also, oral cancers serve to cause or exacerbate periodontal disease and vice versa.

Oballe et al’s study discovered that rats exposed to the carcinogen 4-Nitroquinoline 1-oxide (4-NQO) developed OSCCs, indicating a significant amount of spontaneous alveolar bone loss. It contrasted with the group receiving treatment, which led to the non-development of OSCC, and the control group, which was not given 4-NQO. Consequently, the occurrence of squamous cells with malignant sites in their oral cavities may correlate with an increase in their alveolar bone loss.⁴⁹ Pushalkar et al discovered that, in the same participants, the pathogenic microbial communities at tumour sites tended to be more distinct than those at non-tumour sites.⁵⁰ It has been proposed that this microbial shift causes persistent inflammation in cancer locations and may aid in spreading cancer to nearby locations. This illustrates the correlation between the two illnesses and their causes. The alterations of the mouth milieu may provide circumstances conducive to the emergence of periodontal disease, oral cancers, or their combination. Interleukin 8 (IL-8) protein levels were higher in the saliva of the CA-with-P, CA-without-P, and P groups than in the C group.

Chemotactic cytokines, such as IL-8, stimulate the recruitment of inflammatory cells and increase the severity of periodontal deterioration and progression of cancers. They are produced by several cell types, including mast cells, fibroblasts, endothelial cells, macrophages, and lymphocytes that can induce immune response.⁵¹ Interestingly, this result is in agreement with studies of severe periodontal disease, illustrated by higher mean PPD at sites, which may correlate with higher salivary IL-8 levels.^52,53 Neoplastic patients have higher levels of IL-8 than individuals with normal tissues. The role of IL-8 in the invasion and migration of tumour cells is crucial for tumour growth and metastasis.⁵⁴ The main mediator of oral cancer-related systemic alterations is IL-8.⁵⁵ Cancer tissues also show an over-expression of IL-8. It has also been linked to the metastatic phenotype and highly invasive capacity of cancer cells.^56,57 Their capacity to draw in leukocytes and intensify or prolong the inflammatory response, has significant effects on periodontium illnesses like chronic periodontitis developing with tissue loss. Chronic inflammation is also becoming more recognised as a major factor in the development of tumours. Secreted substances like chemokines play a major role in mediating interactions between cancer cells and their environment and assist in augmenting the phenotypes of malignancy in oral and other cancers. IL-8 cytokines have the biological and biochemical process of chemokine acting in host-microbiome interactions in the pathophysiology of periodontitis and oral cancer.³²

The CA-with-P, CA-without-P, and P groups had significantly higher levels of salivary NF-κB than the C group. Nuclear factor kappa B (NF-κB) regulates cytokine expression through numerous mechanisms. Abnormal NF-κB activity can lead to unregulated expression of interleukin in cancer metastasis and periodontal tissues.⁵⁷ In mammary cancer cells, NF-κB stimulates the transcription of numerous genes encoding cytokines, receptors, and growth factors that lead to the formation and growth of cancers.⁵⁸ Nuclear factor kappa B (NF-κB)’s constitutive activity correlates to aggressive cancer types, with its transcription factors linked to cancer formation and growth.⁵⁸ Furthermore, NF-κB regulates IL-8 gene expression through signals, such as lipopolysaccharide and oxidative stress, resulting in optimum gene activation in cancer cells.^56,58 Timing of NF-κB activation affects transcriptional responses to stimuli. NF-κB activation enhances inflammatory gene transcription, but prolonged activation changes the epigenome and affects a wider variety of genes.⁵⁹ NF-κB signalling activates the family transcription factors, which bind to pro-inflammatory gene promoter regions to directly inhibit RELA and RELB function. This negative feedback loop enhances our knowledge of NF-κB signalling and offers an opportunity for treatment.⁶⁰ NF-κB signalling has a key role in inflammatory reactions. Its function can be broadened to include a range of signalling pathways, biological processes, illness in humans, and therapeutic possibilities. The pathological and physiological conditions of NF-κB signalling, besides its complex roles in immune modulation, inflammation, and the malignant environment.⁶¹

In terms of the anatomical sites of the tumour, the CA-with-P group had a higher percentage of oral (A) and oropharyngeal (B) cancers than the CA-without-P group. Furthermore, oral and oropharyngeal cancer patients had higher periodontitis severity than parapharyngeal cancer patients. This could be because easier access to the oral region allows for earlier identification and diagnosis, which prevents cancer progression during oral examination.^62,63 Pharyngeal cancers closely correlate with these areas as they have more blood and lymph vessels, are challenging to evaluate, and can affect the tumour’s prognosis and progression in addition to making detection and treatment more challenging.⁶⁴

There is a strong correlation between cervical metastases and parapharyngeal carcinoma, with an incidence of 50–70%.⁶⁵ However, due to its greater tumour dissemination, besides being located in regions difficult to visualise and diagnose.⁶⁶

In head and neck squamous cell carcinomas, the oral and the oropharyngeal regions are more commonly affected.^67,68 These regions are more affected in periodontitis patients as well as pathogenic periodontal microbiomes, such as Fusobacterium nucleatum and Porphyromonas gingivalis, are isolated from these sites. Apart from that, bacterial infections are known contributors to the development, advancement, and promotion of cancers.⁴⁰ There are molecular pathways by which periodontal infections contribute to the development of mouth cancer. Pathogens actively interact with the host epithelium through a distinct molecular communication process to acquire cancer characteristics. Additionally, they indirectly promote the development of cancer by creating a pro-inflammatory environment and releasing carcinogenic substances. Cancer development correlates with the functional qualities of the oral microbial population rather than its compositional properties.⁴¹ The results of Shiga et al’s study indicate that the frequency of gene hypermethylation and allelic loss varies significantly across oral and pharyngeal malignancies. Although all of these tumours are identified as squamous cell carcinomas, the carcinogenesis process may change for tumours that are situated in different regions of the head and neck. Tumours in the pharynx arise from the loss of function of tumour suppressor genes caused by allelic loss, whereas carcinogenesis in the oral cavity is caused by the loss of function due to the methylation of those genes’ promoter regions.⁶⁹ Moreover, in underdeveloped nations, the likelihood of oral cancer is higher than that of pharyngeal cancer.⁷⁰ Cancer is exacerbated by persistent inflammation as in periodontal disease.⁷ Carcinogenic consequences are exacerbated by dysbacteriosis in periodontal disease.⁷¹ All of these point to an apparent correlation between periodontal disease and oral, oropharyngeal, and parapharyngeal cancers. Additionally, it implies a causal correlation between oral cancers and periodontitis severity. The influence of perio-pathogens on periodontal diseases and oral, oropharyngeal, and parapharyngeal cancer can describe the pathophysiology of oral cancers, and emphasise the oral hygiene required to prevent oral diseases, particularly oral cancers. The limitation of this study includes a lack of microbiological and biomolecular examinations owing to the need for different sample types and higher costs. The small sample size can result in a decent percentage of oral cavity (A) and oropharyngeal (B) cancers in the P group and the CA-without-P group. The CA-with-P group had slightly lower incidences of parapharyngeal (C) cancers than the CA-without-P group lacks longitudinal data because it requires more time for the study.

Conclusion

The primary finding of this study is a significant correlation between periodontitis with malignancies in the oral, oropharyngeal, and parapharyngeal regions as evidenced by higher levels of salivary IL-8 and NF-κB. This study assessed the mechanisms underlying the correlation between these two disorders, as elucidated by higher levels of salivary IL-8 and NF-κB when compared to controls. They positively and significantly correlate with the increase of clinical periodontal parameters compared with controls. The present evidence strongly suggests that periodontal disease may be an indicator of oral cancers. Owing to the periodontitis severity brought on by perio-pathogens, carcinogenesis can occur elsewhere.³⁸ It typically manifests outside the gingiva, most frequently in the oral cavity and the oropharyngeal region.⁴¹ A microenvironment that facilitates the advancement of cancer is created by periodontal disease-related phenotypic changes and functional abnormalities in the periodontium’s immunological (IL-8, NF-κB) and stromal components.^56,58 However, the tendency to recruit leukocytes and prolonged the process of inflammation may contribute to the advancement of oral illnesses such as chronic periodontitis, causing tissue damage. Furthermore, persistent inflammation is a significant factor in tumour growth. Secreted substances such as chemokines have an important role in the interaction between tumour cells and their surrounding environment, leading to the phenotype of tumours in oral and other malignancies.³² Interleukin 8 (IL-8) plays an important role in carcinogenesis as well as in immune and inflammatory response regulation. Its expression may be deregulated in cancer metastasis due to the aberrant activity of NF-κB.^54,57 Nuclear factor kappa B (NF-κB) transcription factors participate in the development and growth of cancer.⁵⁸ NF-κB signalling has a crucial role in physiological and pathological states, as well as its complex roles in immunological control, inflammation, and the cancerous microenvironment.⁶¹ Thus, the inflammatory environment in periodontal disease is perfect for the seeding, migration, proliferation, and escape of cancer cells. Therefore, periodontitis patients are more susceptible to malignancies affecting the oral, oropharyngeal, and parapharyngeal regions, and periodontitis severity may serve as a predictor of oral, oropharyngeal, and parapharyngeal cancers.