Biochemical markers in saliva in patients with oral cancer

SUMMARY Head and neck cancers account for 3% of all human cancers and are mainly localized in the oral cavity. Early detection of cancer is extremely important for reducing mortality and morbidity from this disease. In addition to clinical trials and histopathological findings, in the last two decades, with technological development, more specific and sensitive methods have been used in the analysis of salivary markers. The aim of this study was to present a wide range of analyzed markers in saliva: different protein markers (total proteins, albumin, defensins, staterin, cystatins), epithelial and molecular markers (CA125, CA19-9, TPA, CEA, CYFRA 21-1, CD44), enzymes (LDH, ALP, MMP, SOD), cytokines (IL-6, IL-8, IL-1 β , TNF- α ), oxidative stress markers (8-OHdG, MDA), non-enzymatic antioxidants (glutathione, uric acid, albumin, vitamin C and E) etc. Collecting saliva is simple and painless for the patient, it does not require expensive equipment or specially trained staff, and it is possible to take saliva several times and in unlimited quantities. Extensive research that is increasingly being done with modern techniques indicates that saliva may be useful for early detection of the disease in the near future.


INTRODUCTION
Head and neck cancers account for 3% of all human cancers and are mainly localized in the oral cavity (48%). The most common (90%) are squamous cell carcinomas, with an incidence of over 300,000 cases per year worldwide. The tongue (over 40%) and floor of the mouth are the most common localizations of this tumor in the oral cavity, and it is less often localized in the area of the gingiva, buccal mucosa, labial mucosa and hard palate. The most important risk factors for the development of oral cancer are smoking, alcohol, tobacco smoke agents, human papilloma virus and others [1,2,3].
Early detection of oral cancer is extremely important for reducing mortality and morbidity from this disease. The diagnosis is made on the basis of clinical analyzes and histopathological findings after taking the biopsy. Recently, more work is being done on the potential use of non-invasive methods -"liquid biopsy" -for the detection of diagnostic and prognostic biomarkers in body fluids [4,5]. Identification and characterization of markers in saliva would help in the diagnosis and monitoring of patients with pre-malignant and malignant lesions of the oral cavity, patients in the postoperative procedure or the application of adequate therapy [6][7][8][9]. In the monitoring of oral cancers, the term "salivaomics" has been introduced, for a wide range of technologies that investigate different types of molecules found in saliva [10].
The aim of this study is a detailed presentation of various markers that were analyzed in the saliva of patients with oral cancer.

PROTEIN MARKERS IN SALIVA
Protein markers in the saliva of patients with oral cancer are analyzed individually or in groups, for the purpose of early detection of the disease and timely application of appropriate therapy.
In the saliva of patients with oral cancer, the concentration of total proteins was increased compared to the control group [11,12]. Other studies have shown that saliva of untreated patients has an increased concentration of total protein compared to treated patients with oral cancer and patients with precancerous lesions [13]. The authors believe that this is a consequence of locally increased protein synthesis. Sanjay et al. came to similar results in cancers with different degrees of differentiation, but the difference in total protein concentration was not statistically significant [14]. The opposite results were obtained, which indicate that the total proteins are reduced in the saliva of patients with oral cancer [15].
In the recent years, with technological developments, more specific and sensitive methods have been used to identify total salivary proteins or peptides, known as salivary proteomes. About a thousand proteins are present in saliva, with different roles in metabolic processes, immune regulation, cell adhesion, communication, etc. [16,17].
Albumins are plasma proteins that are synthesized in the liver. The roles of albumin are maintaining oncotic pressure, regulation of blood pH, transport of various substances, but also antioxidant protection. Our studies indicate a significant decrease in the concentration of albumin in the saliva of patients with oral cancer compared to the control group [18]. This can be explained by the "consumption" of this antioxidant in neutralizing free radicals, which are excessively produced in these pathological conditions. In other studies, the opposite results were obtained [19].
The best known tumor suppressor gene is p53 (16-20 kb DNA) is localized on human chromosome 17. Mutation, inactivation and deletion of the p53 gene are also involved in the pathogenesis of oral tumors. Increased expression of the p53 gene has been found in saliva in oral squamous cell carcinoma. A high percentage (71 %) of tumor-specific mutations in the p53 gene has also been demonstrated in these patients [20].
Defensins are salivary peptides that exhibit antimicrobial activity and are extremely important in maintaining oral health [21]. They are proven in granular leukocytes -neutrophils, so they are called human neutrophil defensins. The concentration of defensin-1 in the saliva of patients with oral squamous cell carcinoma and other oral diseases (lichen planus, leukoplakia, inflammation) is significantly higher compared to saliva of the control group. This is in accordance with the fact that during many diseases in the oral cavity, a large number of neutrophils migrate from the blood through gingival sulcus to the oral environment [22].
Staterin is an acidic salivary protein that prevents the deposition of calcium phosphate in the excretory ducts of the salivary glands and regulates solubility of tooth enamel [21,23]. In the saliva of patients with oral cancer, the concentration of staterin is reduced, and thus its function in oral cavity is reduced too [24].
Cystatins are proteins by chemical structure and inhibitors of the enzyme cysteine proteases by function. Cystatin SA-I, which has 14 kDa, has been detected in the saliva of patients with oral squamous cell carcinoma. This protein is more pronounced in the saliva of patients before treatment compared to the saliva of treated patients, so it may be a useful biomarker for patients with oral squamous cell carcinoma [25].
Epidermal growth factor (EGF) is a protein that plays a significant role in maintaining the homeostasis of the oral mucosa and mucosa of the upper gastrointestinal tract. It also promotes wound healing in the oral environment.
Smoking and alcohol consumption have been shown to reduce the level of salivary EGF, which contributes to the development of oral carcinogenesis. In the saliva of patients with oral cancer, the concentration of EGF is reduced, and thus the possibility of renewal of the epithelium of the oral mucosa in these patients is reduced [26,27].

SIALIC ACID, EPITHELIAL MARKERS, ENZYMES
Sialic acid is located at the terminal end of glycoproteins and glycolipids and plays a significant role in cell to cell interactions and development of cell adhesion important in malignant transformation [28]. Sialic acid concentration is increased in saliva of well-differentiated squamous cell carcinomas compared to poorly differentiated carcinomas [14]. Other authors have found its increased concentration relative to pre-malignant lesions in healthy individuals [11,29]. After radiotherapy of patients with oral cancer, the level of sialic acid in the saliva is reduced, so it can be said that sialic acid is a sensitive tumor marker [30].
Epithelial markers (CA125, CA19-9, tissue polypeptide antigen, carcino-embryonic antigen, CYFRA 21-1) have an increased concentration in the saliva of patients with oral squamous cell carcinoma. In particular, the three analyzed markers (CYFRA-21, tissue polypeptide antigen, CA-125) were significantly increased (by 400 %), while for other markers no statistical significance was found [6]. Similar results were obtained by other authors for CYFRA 21-1 [31]. Analysis of these tumor markers in the saliva of patients with oral cancer may be suggested as an aid, rather than as a substitute, for other well-established diagnostic methods.
Molecular marker, the CD44 protein, can be converted to a soluble form by the action of proteases. It is increased in the saliva of patients with oral cancer compared to the control group. A perfect correlation between salivary CD44 molecules and grade and the degree of aggressiveness of the malignant lesion has been demonstrated. There is also a high statistically significant difference between patients with oral cancer and patients with pre-malignant lesions. This is indicated by the fact that the concentration of salivary CD44 between 19.2 and 20.4 ng/mL may indicate malignant transformation of lesions of the oral mucosa [32].
Enzymes, responsible for playing role in metabolic processes in cells, were also analyzed in saliva. The activity of lactate dehydrogenase and alkaline phosphatase is increased in the saliva of patients with oral squamous cell carcinoma [33,34]. Merza et al. have demonstrated increased activity of these enzymes in the serum of patients with this disease [35]. The authors believe that the release of intracellular enzymes is increased from pathologically altered cells, rather than a consequence of increased biosynthesis. Matrix metalloproteinases (MMPs) are enzymes involved in the pathogenesis of oral cancer. Unregulated MMP activity in tumor tissues is one of the main factors of protein destruction (collagen, elastin, fibronectin). Increased activity of MMP-2 and MMP-9 has been demonstrated in the saliva of patients with oral squamous cell carcinoma (OSCC) [33,36]. Peisker et al. demonstrated significantly increased MMP-9 activity in patients with OSCC compared to the control group (19.2 %), whether it was the first diagnosis or recurrence. The sensitivity of this marker was 100 % and the specificity 26.7 % [37].

CYTOKINES AND MARKERS OF OXIDATIVE STRESS
Cytokines represent a family of soluble, low molecular weight proteins or glycoproteins, which function as mediators and modulators of the immune response, inflammation, hematopoiesis, and development of malignant tumors. The concentration of interleukin-6 (IL-6), IL-8 and tumor necrosis factor (TNF-α), which act as promoters in the process of carcinogenesis, was increased in the saliva of patients with oral cancer [38]. The most commonly determined cytokine in the saliva of patients with oral squamous cell carcinoma is IL-6 [39]. Brailo et al. have demonstrated increased concentrations of salivary IL-6 and IL-1β in patients with oral cancer compared to patients with leukoplakia. They leave the possibility to examine whether these cytokines are markers of malignant transformation of leukoplakia before oral cancer becomes clinically evident [40]. Other studies have indicated an increased content of IL-1, IL-6, TNF-α in the saliva of subjects with oral squamous cell carcinoma compared to patients with dysplastic oral lesions and control groups of subjects. Because of the above, salivary cytokines provide useful information on the behavior of epithelium in carcinogenesis and may be potential biochemical markers of oral cancer [41].
In the pathogenesis of oral cancer nowadays free radicals and oxidative stress are given increasing importance. Free radicals of oxygen and nitrogen lead to oxidative modification of proteins, lipids, DNA of oral tissue cells, which can result in their malignant alteration [42]. The most important biomarker of the degree of oxidative DNA damage is 8-hydroxy-deoxyguanosine (8-OHdG), the concentration of which is increased in the saliva of patients with oral squamous cell carcinoma [43]. The end product of lipid peroxidation is malondialdehyde (MDA). By analyzing the concentration of MDA, we showed that in the saliva of patients with periodontal disease there is an increase in its content compared to the group of healthy subjects [44]. Other authors have obtained similar results in the saliva of patients with oral squamous cell carcinoma [45].
In addition, toxic components from tobacco smoke affect the change in the antioxidant capacity of saliva [46]. Decreased antioxidant enzyme activity results in incomplete elimination of H 2 O 2 from the oral environment, which reacts with other radicals and molecules to form much more reactive free radicals, which oxidatively damage biomolecules such as DNA, and that can lead to malignant transformation and oral cancer [47,48]. The concentration of non-enzymatic antioxidants in saliva, such as glutathione, is also reduced. The authors explain this by the interaction of tobacco smoke aldehydes and SH groups of glutathione, when nonfunctional conjugates are formed [11,46]. It is especially interesting to analyze the concentration of uric acid, as the main non-enzymatic antioxidant, which participates with about 70% in the total antioxidant capacity of saliva. Our and other studies showed that in the saliva of patients with oral cancer, who were smokers, the concentrations of uric acid and albumin were significantly reduced, compared to the group of healthy subjects. These results can be explained by the increased "consumption" of these antioxidants in neutralizing free radicals [18,49]. Decreased concentrations of vitamins E and C in the saliva of patients with increase of histological grade of oral cancer have also been demonstrated [50]. From the above, it can be concluded that the analysis of the antioxidant capacity of saliva can be useful for improving preventive measures in the development of oral cancer, so, as in the case of periodontitis, the use of various antioxidants is recommended.

CONCLUSION
Based on this review, it was determined that biochemical composition of saliva changes in patients with oral cancer. Saliva analyzes in these patients have advantages and disadvantages. Saliva collection is simple and painless for the patient, it does not require expensive equipment or specially trained staff, which certainly goes in favor of cost-effectiveness. It is also possible to take saliva several times and in unlimited quantities. However, some of the present problems cannot be ignored. Individual biomarkers proven in saliva are not sensitive and specific enough to meet stringent diagnostic criteria. There is also the problem of extremely high saliva viscosity, due to the presence of mucopolysaccharides and mucoproteins, which can interfere with the analytical procedure. Despite these limiting circumstances, extensive research, increasingly done with modern techniques, indicates that saliva may be useful for early detection, diagnosis and monitoring of applied therapy for oral cancer in the near future.