Saliva is secreted from the salivary glands and has multiple functions, including mouth cleaning and protection, antibacterial effects and digestion. With the rapid advancement in salivaomics, saliva is well recognized as a pool of biological markers. Saliva, as a non-invasive and safe source, could be a substitute for blood in the diagnosis and prognosis of diseases. This review summarizes the latest advancements in saliva-related studies and addresses the potential value of saliva in the early diagnosis of oral diseases, such as dental caries and periodontal disease, as well as cancer, diabetes and other systemic disorders. Saliva biomarkers range from changes in the biochemical indices of DNA, RNA and proteins to the diversification of microbiota structures. This study integrates data reported in the recent literature and discusses the clinical significance and prospects for the application of saliva in the early diagnosis of diseases, translational medicine and precision medicine.

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Saliva in the diagnosis of diseases.

Tissue necrosis factor-α (TNF-α) and complement component 3 (C3) are two well-known pro-inflammatory molecules. When TNF-α is upregulated, it contributes to changes in coagulation and causes C3 induction. They both interact with receptors on platelets and erythrocytes (RBCs). Here, we look at the individual effects of C3 and TNF-α, by adding low levels of the molecules to whole blood and platelet poor plasma. We used thromboelastography, wide-field microscopy and scanning electron microscopy to study blood clot formation, as well as structural changes to RBCs and platelets. Clot formation was significantly different from the naive sample for both the molecules. Furthermore, TNF-α exposure to whole blood resulted in platelet clumping and activation and we noted spontaneous plasma protein dense matted deposits. C3 exposure did not cause platelet aggregation, and only slight pseudopodia formation was noted. Therefore, although C3 presence has an important function to cause TNF-α release, it does not necessarily by itself cause platelet activation or RBC damage at these low concentrations. We conclude by suggesting that our laboratory results can be translated into clinical practice by incorporating C3 and TNF-α measurements into broad spectrum analysis assays, like multiplex technology, as a step closer to a patient-orientated, precision medicine approach.

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The inflammatory effects of TNF-α and complement component 3 on coagulation

Oral cancer is the sixth most common cancer type in the world, and 90% of it is represented by oral squamous cell carcinoma (OSCC). Despite progress in preventive and therapeutic strategies, delay in OSCC diagnosis remains one of the major causes of high morbidity and mortality; indeed the majority of OSCC has been lately identified in the advanced clinical stage (i.e., III or IV). Moreover, after primary treatment, recurrences and/or metastases are found in more than half of the patients (80% of cases within the first 2 years) and the 5-year survival rate is still lower than 50%, resulting in a serious issue for public health. Currently, histological investigation represents the "gold standard" of OSCC diagnosis; however, recent studies have evaluated the potential use of non-invasive methods, such as "liquid biopsy," for the detection of diagnostic and prognostic biomarkers in body fluids of oral cancer patients. Saliva is a biofluid containing factors such as cytokines, DNA and RNA molecules, circulating and tissue-derived cells, and extracellular vesicles (EVs) that may be used as biomarkers; their analysis may give us useful information to do early diagnosis of OSCC and improve the prognosis. Therefore, the aim of this review is reporting the most recent data on saliva biomarker detection in saliva liquid biopsy from oral cancer patients, with particular attention to circulating tumor DNA (ctDNA), EVs, and microRNAs (miRNAs). Our results highlight that saliva liquid biopsy has several promising clinical uses in OSCC management; it is painless, accessible, and low cost and represents a very helpful source of diagnostic and prognostic biomarker detection. Even if standardized protocols for isolation, characterization, and evaluation are needed, recent data suggest that saliva may be successfully included in future clinical diagnostic processes, with a considerable impact on early treatment strategies and a favorable outcome.

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Salivary Biomarkers for Oral Squamous Cell Carcinoma Diagnosis and Follow-Up: Current Status and Perspectives.

Background Oral squamous cell carcinoma is the most common manifestation of malignancy in the oral cavity. Adjuncts are available for clinicians to evaluate lesions that seem potentially malignant. In this systematic review, the authors summarized the available evidence on patient-important outcomes, diagnostic test accuracy (DTA), and patients' values and preferences (PVPs) when using adjuncts for the evaluation of clinically evident lesions in the oral cavity. Types of Studies Reviewed The authors searched for preexisting systematic reviews and assessed their quality using the Assessing the Methodological Quality of Systematic Reviews tool. The authors updated the selected reviews and searched MEDLINE, Embase, and the Cochrane Central Register of Controlled Trials to identify randomized controlled trials and DTA and PVPs studies. Pairs of reviewers independently conducted study selection, data extraction, and assessment of the certainty in the evidence by using the Grading of Recommendations Assessment, Development and Evaluation approach. Results The authors identified 4 existing reviews. DTA reviews included 37 studies. The authors retrieved 7,534 records, of which 9 DTA and 10 PVPs studies were eligible. Pooled sensitivity and specificity of adjuncts ranged from 0.39 to 0.96 for the evaluation of innocuous lesions and from 0.31 to 0.95 for the evaluation of suspicious lesions. Cytologic testing used in suspicious lesions appears to have the highest accuracy among adjuncts (sensitivity, 0.92; 95% confidence interval, 0.86 to 0.98; specificity, 0.94; 95% confidence interval, 0.88 to 0.99; low-quality evidence). Conclusions and Practical Implications Cytologic testing appears to be the most accurate adjunct among those included in this review. The main concerns are the high rate of false-positive results and serious issues of risk of bias and indirectness of the evidence. Clinicians should remain skeptical about the potential benefit of any adjunct in clinical practice.

Adjuncts for the evaluation of potentially malignant disorders in the oral cavity: Diagnostic test accuracy systematic review and meta-analysis—a report of the American Dental Association

Each year approximately 6,000 new cases of head and neck cancer are registered in Poland. Human papillomavirus (HPV) and Epstein-Barr virus (EBV) have been associated with tumour formation. Cytokines have been shown to play an important role both in inflammation and carcinogenesis and they can be detected in saliva and serum with ELISA assays. Salivary biomarkers may be used as markers of early cancer detection. The aim of this study was the analysis of the serum and salivary levels of IL-10, TNF-α, TGF-β and VEGF in patients with oropharyngeal cancer and in healthy individuals. The level of these biomarkers was also analyzed in HPV- and EBV-related cases. The study involved 78 patients with histopathologically confirmed oropharyngeal squamous cell carcinoma and 40 healthy controls. Serum and salivary levels of IL-10, TNF-α, TGF-β and VEGF were analyzed both in patients and in healthy individuals by ELISA method using Diaclone SAS commercially available kits (France). EBV DNA was detected by the nested PCR for amplification of EBNA-2. HPV detection and genotyping was performed using the INNO-LiPA HPV Genotyping Extraassay (Innogenetics N. V, Gent, Belgium). The obtained results were subjected to statistical analysis using Mann–Whitney and Kruskal Wallis tests. Test values of p < 0.05 were considered statistically significant. The level of tested cytokines was higher in patients than in controls both in serum (IL-10: 2.3 pg/ml vs 1.65 pg/ml, p = 0.0003; TGF-β: 11.3 ng/ml vs 7.8 ng/ml, p = 0.0005; VEGF: 614 pg/ml vs 210 pg/ml, p = 0.0004; TNF-α: 15.0 ng/ml vs 12.90 ng/ml, p = 0.1397) as well as in saliva (IL-10: 5.9 pg/ml vs 2.5 pg/ml, p = 0.00002; TGF-β: 24.1 ng/ml vs 14.8 ng/ml, p = 0.00002; VEGF: 4321 pg/ml vs 280 pg/ml, p = 0.0000; TNF-α: 23.1 ng/ml vs 11.3 ng/ml, p = 0.00002). EBV DNA was detected in 51.3 % of patients and 20 % of controls, HPV DNA was present in 30.8 % of patients and 2,5 % of controls. The level of IL-10 was statistically higher in patients infected with EBV, HPV and co-infected with EBV/HPV. The level of TNF-α was significantly higher in patients infected with EBV, while TGF-β in patients with HPV infection and EBV/HPV co-infection. Detection of salivary cytokines may be very helpful in early diagnosis, treatment and prognosis of OSCC.

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Salivary and serum IL-10, TNF-α, TGF-β, VEGF levels in oropharyngeal squamous cell carcinoma and correlation with HPV and EBV infections

Background: Oral squamous cell carcinoma (OSCC) is an important malignancy throughout the world; early detection is an important criterion for achieving high cure rate. Out of the many reported markers for OSCC, this study validated the efficacy of tumor necrosis factor-α (TNF- α) in differentially diagnosing premalignant oral lesions and OSCC. Also, the study aimed to correlate the levels of salivary and serum TNFα with clinicopathologic factors. Materials and Methods: A prospective experimental laboratory study was designed. Serum and salivary samples from 100 subjects in each group of healthy control, premalignant disease (PMD) and OSCC were collected for the study following appropriate exclusion and inclusion criteria. Serum and salivary level of TNF-α was analysed by enzyme linked immunosorbent assay. The data obtained were subjected to appropriate statistical analysis. Results: Increased level of both serum and salivary TNF- α was observed in OSCC subjects compared to healthy control and PMD group. Receiver operator characteristic curve analysis and area under curve values showed high specificity and sensitivity for salivary TNF-α in differentiating OSCC from PMD and healthy controls. There was significant increase in TNF- α level in moderately and poorly differentiated lesion compared to well differentiated lesion and in stage IV of clinical stage. A positive correlation was observed only with histological grading of OSCC and TNF- α. Conclusions: Salivary TNF-α is proved to be superior for detecting OSCC. Increase in TNF-α with histological grading and clinical staging suggests a role in prognosis.

http://koreascience.or.kr:80/article/JAKO201429765166808.pdf

Association of serum and salivary tumor necrosis factor-α with histological grading in oral cancer and its role in differentiating premalignant and malignant oral disease.

The development of low- or non-invasive screening tests for cancer is crucial for early detection. Saliva is an ideal biofluid containing informative components for monitoring oral and systemic diseases. Metabolomics has frequently been used to identify and quantify numerous metabolites in saliva samples, serving as novel biomarkers associated with various conditions, including cancers. This review summarizes the recent applications of salivary metabolomics in biomarker discovery in oral cancers. We discussed the prevalence, epidemiologic characteristics, and risk factors of oral cancers, as well as the currently available screening programs, in India and Japan. These data imply that the development of biomarkers by itself is inadequate in cancer detection. The use of current diagnostic methods and new technologies is necessary for efficient salivary metabolomics analysis. We also discuss the gap between biomarker discovery and nationwide screening for the early detection of oral cancer and its prevention.

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Salivary Metabolomics for Oral Cancer Detection: A Narrative Review

Pemphigus vulgaris (PV) is a chronic, autoimmune, intraepidermal blistering disease of the skin and mucous membranes. The initial clinical manifestation is frequently the development of intraoral lesions, and later, the lesions involve the other mucous membranes and skin. The etiology of this disease still remains obscure although the presence of autoantibodies is consistent with an autoimmune disease. These antibodies are targeted against the adhesion proteins of keratinocytes, leading to acantholysis (disruption of spinous layer, leading to intraepidermal clefting) and blister formation. Because only oral lesions are present initially, the chances of misdiagnosing the disease as another condition are increased, leading to inappropriate therapy. In this article, we report a case of PV with only oral manifestations in a 36-year-old male.

Oral pemphigus vulgaris: A case report with direct immunofluorescence study

Objective: Squamous Cell Carcinoma is almost always preceded by potentially malignant disorders in the oral cavity before malignant transformation. Characterization of  8-OHdG from the saliva offers a relatively non-invasive, simple and efficient methodology for monitoring oxidative stress in subjects of Premalignant oral disorders (PMOD) and Oral Squamous Cell Carcinoma (OSCC). Hence the aim of the current study is to estimate the levels of salivary 8-hydroxydeoxyguanosine (8-OHdG) as a potential DNA Damage Biomarker in OSMF and OSCC patients in comparison to healthy individuals to assess disease progression from potentially malignant oral disorder to frank malignancy. Materials and Methods: The study was conducted among 90 patients [Oral Squamous cell carcinoma (n=30) and Oral Submucous Fibrosis (n=30) and healthy gender and age matched controls (n=30)]. 4ml of unstimulated saliva was collected from each of the subjects and was subjected to Sandwich ELISA for the quantification of salivary 8-OHdG. Statistical analysis was done using ANOVA, and p value was set at ≤0.05. Results: The mean age of OSCC patients were 56.8±11.8 years. Smoking was the most prevalent adverse habit among this group (66.6%) followed by Smokeless tobacco chewers (40%). The mean age of OSMF patients was 46.2± 9.8 years. Smokeless tobacco was the most predominant habit among the OSMF patients (83.33%) followed by smoking (33.33%). The mean OHdG levels among the controls was 6.59±1.47 (ng/dl) and almost doubled in patients of OSMF 13.89±1.96(ng/dL) and further raised in OSCC patients 19.96 ± 2.11 (ng/dL). These levels showed a highly significant difference (p <0.0001) in mean on comparison by using one-way ANOVA. Pearson correlation between the groups were also statistically significant (p=0.000). Conclusion: There were significant differences in the concentration of salivary 8-OHdG between healthy controls, OSMF, and OSCC patients. Hence, 8-OHdG can be used as a novel biomarker of DNA damage to assess disease progression.

http://journal.waocp.org/article_89223_db2e91652404cdfeadd8f4ac663c2288.pdf

Estimation of Salivary 8-Hydroxydeoxyguanosine (8-OHdG) as a Potential Biomarker in Assessing Progression towards Malignancy: A Case-Control Study.

Background: Exfoliated oral cancer cells in saliva samples from patients with oral squamous cell carcinoma (OSCC) can be used to determine the incidence and type of mutations of the p53 tumor suppressor gene. The purpose of this study was to identify C-deletion mutation in exon 4 codon 63 of p53 gene in the saliva of OSCC patients by polymerase chain reaction (PCR). Materials and Methods: Saliva samples of 20 newly histopathologically diagnosed OSCC patients and 5 healthy volunteers were subjected to isolation of the total genomic DNA and PCR amplification for C-deletion on exon 4 of p53 gene. The resulting products were resolved by agarose gel electrophoresis, viewed and photographed on ultraviolet-transilluminator. Results: The relationship between the frequencies of genetic alterations was assessed by Chi-square test. Differences with values of P Conclusion: The study concluded a 100% presence of C-deletion mutation in exon 4 codon 63 of p53 in the saliva of OSCC patients. This study suggests that detection of mutation in exon 4 codon 63 of p53 by PCR is a fast, reliable, accurate, and sensitive molecular method for OSCC diagnosis.

Rajkumar Krishnan

Papers

Association of serum and salivary tumor necrosis factor-α with histological grading in oral cancer and its role in differentiating premalignant and malignant oral disease.

Salivary Metabolomics for Oral Cancer Detection: A Narrative Review

Oral pemphigus vulgaris: A case report with direct immunofluorescence study

Estimation of Salivary 8-Hydroxydeoxyguanosine (8-OHdG) as a Potential Biomarker in Assessing Progression towards Malignancy: A Case-Control Study.

C-deletion in exon 4 codon 63 of p53 gene as a molecular marker for oral squamous cell carcinoma: A preliminary study.