A biomarker is defined as ‘a biological molecule found in blood, other body fluids, or tissues that is a sign of a normal or abnormal process, or of a condition or disease’ by the National Cancer Institute 1 . The biomarker, also called a molecular marker, has a wide range of applications in diagnosis, monitoring of treatment, and the prognosis of a disease or condition. Despite attempts to classify biomarkers of cancer, a consensus has not been established. Mishra and Verma 1 have suggested that the classification of biomarkers can be based on the disease state, biomolecules, or other criteria.(Fig. 1)
A peer-review analysis by the World Health Organization International Agency for Research on Cancer (WHO IARC) reported that the global estimated rate for oral cavity cancer was 2.7 per 100,000 in 2012 2,3 . Oral cancers are also the eighth most common causes of cancer-related deaths worldwide 4 . Oral squamous cell carcinoma (OSCC) accounts for over 90% of oral cancers and is considered to be a rising global public health issue because of its high incidence and low survival rate 5,6 .
In the efforts to reduce OSCC-related mortality, enhancing and innovating screening and early detection technologies has been suggested as the most effective and fastest developing strategy 7 . In this area, the liquid biopsy came up as a noninvasive diagnostic technique that is based on the detection of tumor markers in body fluids. In addition to blood, saliva has a role as an auxiliary tool in oral cancer diagnosis 8 . Furthermore, recent studies have revealed that saliva sampling can be a more effective method of detecting specific OSCC biomarkers 9 .
Recently, diagnostic markers are the focus of our clinical and experimental studies. A diagnostic cancer marker can be specific to stage, tissue, relapse, follow-up, or age 9 , and present at any stage during cancer development. This review article introduces an updated list of reported OSCC salivary biomarkers up until 2019 and discusses the current clinical application of salivary biomarkers.
Whole saliva also contains a variety of non-organic and organic substances from the serum, gingival crevicular fluid, as well as oral microorganisms and their products10,11. In addition to a diversity of biomarkers for many diseases, saliva’s collection is noninvasive and convenient, and the transportation and storage are easy, therefore saliva sampling is cost-effective and efficient 12 . These advantages demonstrate that saliva is a potential body fluid for laboratory tests compared to serum and tissue samples.
Biomarkers are detected and determined by various molecular techniques. For the genomic biomarkers (including DNA, mitochondrial DNA [mt.DNA], RNA, messenger RNA [mRNA], microRNA [miRNA]), the utilized techniques can be DNA microarrays, polymerase chain reaction (PCR), Southern blot analysis, restriction fragment length polymorphism (RFLP), and cross-linking immunoprecipitation (CLIP). The proteomic biomarkers class includes proteins, peptides, antibodies, and can be analyzed by liquid chromatography, Western blot analysis, protein sequencing, protein arrays, and immunofluorescence. The metabolomics biomarkers (including carbohydrates, enzymes, metabolites, liquids) are determined by liquid chromatography, nuclear magnetic resonance, enzyme assays, and mass spectrometry 11 . One of the earliest developed saliva biomarkers, human papillomavirus (HPV) markers, have been used as the diagnostic biomarker in the cervical cancer screening program and vaccine development 1 .
The identification of reliable salivary biomarkers for the OSCC screening has been enhanced thanks to the easy and noninvasive collection of saliva compared to the drawing of blood 9 . The underlying tissue changes in the disease process can be classified as genomic, proteomic, or metabolomic expression.(Fig. 2) With the development of salivaomics, a lot of researches have been performed and more than 100 potential saliva biomarkers for OSCC have been reported in the literature 13 . Salivary diagnostic has been an attractive potential modality screening, early detection and prognosis evaluation for researchers and clinicians 14 .
The classification of biomarkers can be based on the disease state, biomolecules, or other criteria 1 . Currently, we are paying attention to the screening and early detection of OSCC, and diagnostic markers are in the focus of our clinical and experimental studies. Diagnostic markers may be present at any stage of cancer development.(Table 1) The expression of Eph and/or ephrin are common in various primary tumors, including OSCCs. Among the various biological functions of ephrin and Eph receptors in cancer, their involvement in EFNB2/EphB4 signaling is thought to be associated with angiogenesis, differentiation, and development. Therefore, EFNB2 gene expression is suggested to be a useful biological marker in prognostic evaluation in patients with OSCC 15 . Shpitzer et al. 16 reported that the levels of 8-oxoguanine DNA glycosylase, phosphorylated-Src, and mammary serine protease inhibitor (Maspin) were found to decrease in the saliva of OSCC patient. Several studies reported that interleukin (IL)-6 and IL-8, which are well-known as post-inflammatory cytokines, significantly increase in patients with OSCC and therefore suggesting their value as a diagnostic marker of oral malignant and premalignant lesions 17,18 . Arellano-Garcia et al. 19 reported that both IL-8 and IL-1β were found to significantly increase in OSCC patients.
A diagnostic cancer marker can be specific to tissue, stage, follow-up, relapse, and age. Despite the attempts made to classify cancer biomarkers, a consensus has not been established. However, for the diagnosis and comprehension of the OSCC genomic architecture, more recent efforts have focused on new and noninvasive methods using human saliva sampling, which include proteomic (Table 2)20-25, proteins (Table 3)20,25-36, transcriptomic (Table 4)20,37-41, and metabolomic (Table 5)20,42-44 biomarkers.
Laboratory examination is an essential and high accurate method for disease diagnosis and prognosis. Among the available laboratory test, liquid biopsy is a less invasive method that limits the need for acquiring tissue 45 . In the past 20 years, liquid biopsy has become an essential examination in multiple areas of oncology, based on the detection of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and circulating tumor RNA (ctRNA), proteins, and exosomes 8 . Liquid biopsy samples include blood, urine, saliva, and other bodily fluids such as seminal plasma, pleural effusions, cerebrospinal fluid, sputum, and stool samples 46 .
Saliva has complex components that originate from the major and minor salivary glands, as well as from the oropharynx, gastrointestinal reflux, gingival crevicular fluid, and blood. The analysis of saliva components has been considered an effective method for monitoring the status of health 47 because changes in the compounds that constitute saliva can reflect the physiological and pathological status of the body.
Currently, the most common liquid biopsy is blood. Approximately, 5 to 10 mL of blood is all that is needed for a liquid biopsy. Blood biomarkers are classified as CTCs, ctDNA, ctRNA, proteins, and exosomes, which can be used for differential diagnosis, prognosis, cancer recurrence detection, tumor evolution monitoring, and treatment efficacy in various types of tumors8,48. Circulating cell-free DNA (cfDNA) is released into the bloodstream from apoptotic or necrotic cells. cfDNAs can originate from nonmalignant host cells and tumor cells, thus including ctDNA. There are also authors suggesting that tumor exosomes can play an important role in immune suppression and enhancing tumor development, and plasma exosomes can be the next generation of biomarkers in head and neck cancer progression evaluation8.
There are a lot of methods are for the whole saliva collecting, such as draining, spitting, suction, and swabs49. A variety of commercial devices and methods for collecting saliva from individual glands have also been developed20,49.(Table 6) The clinician should be properly trained on performing saliva sample collection to achieve the best performance and samples. Despite the variety of choices, only one type of collection device should be used in one study50,51. The eligible participants need to be educated and given the appropriate instructions before the collecting procedure. The sample volume needs to be sufficient, and the type of container needs to be prepared beforehand accordingly. Besides, sample labeling and handling protocols must be well-designed and carried out consistently52.
Saliva components can vary or remain stable at age53. It is reported that children and adults also can have differences in the salivary level of a specific protein, peptide, and proteome54. The unstimulated saliva secretion was higher in healthy men compared with women55, indicating gender-dependent secretion. Due to these variables, patients should be categorized into different age groups and gender to minimize statistical errors.
The position of the mouth during saliva collection is important from the basis of salivary glands location and their secretion patterns49. While saliva secreted from the major salivary glands contains common components, the concentrations of each component and some specific contents can vary from one gland to another. On the other hand, components of the saliva from the minor glands mainly include mucins and lipase51.
Besides, saliva components and origin depend on the resting state or stimulated state of the individual. For example, cortisol, alpha-amylase, and secretory IgA levels in the saliva are affected by stimulation49. The complex in the composition of saliva and its dependence on various conditions should be considered and evaluated thoroughly by the investigator. This consistency in sampling procedure is essential to achieve valuable data.(Table 7)
The saliva samples can be kept at room temperature for a maximum of 30 to 90 minutes for the immediate analysis56. Thomadaki et al.10 recommended that the samples are frozen at or below –20ºC immediately after collection, due to the low temperature can slow down the degradation of the salivary proteome. Salivary specimens can also be stored at –80ºC for several years with little or no degradation56. In RNA analysis sampling, an RNase inhibitor should be added in the supernatant fractions before storing it at –80ºC57. Shirtcliff et al.58 suggested that the collection method was an important cause of the unsystematic error. Samples obtained by spitting contain more bacteria than drooling samples, which can affect the analysis results of saliva compounds56.
As with saliva, blood is a complex fluid that contains a wide range of molecular components, including various antibodies, growth factors, enzymes, and hormones. Therefore, blood serum and plasma are the traditional and conventional sources of liquid biopsy and biomarker examination. Although used widely, blood sampling and analysis can often be expensive, problematic, and invasive.
Comparatively, sampling saliva has many advantages over blood, including the following 52 : 1) The saliva sampling procedure is easier and can be self-collected, 2) the procedure is noninvasive, 3) samples are safer to handle and 4) are easier to transport and store because saliva requires simpler manipulation than blood, and 5) the procedure is cost-efficient both for patients and the investigators.
Despite these advantages, the use of saliva sampling as a diagnostic tool is still under controversy. The greatest obstacle in the development of a salivary diagnostic protocol is that while most biomarkers detected in the blood serum are also can be found in saliva, but their levels are so low that they barely can contribute to the diagnosis. For example, the normal level of IgG (5 to 30 mg/mL vs 5 to 30 μg/mL) and IgM (0.5 to 1 mg/mL vs 5 to 10 μg/mL) also have a huge difference in serum and saliva analysis 59 . However, the link between blood and saliva at a molecular level may also suggest that saliva can be a potential alternative to blood- and tissue-based diagnostics.
While there are reports about many saliva biomarkers, the correlation of their levels in the blood and saliva are various, and there are a scarce number of publications on the blood levels of specific biomarkers found in saliva, or how the saliva collection technique affects the quality and diagnosis value of specific biomarkers. Williamson et al. 59 analyzed the correlation of biomarkers among passive drool saliva, filter paper saliva, and plasma samples in healthy adults. The author found that between passive drool and filter paper saliva samples, statistically significant correlations were found among 16 cytokines, including IL1β, IL-1ra, IL-4, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, granulocyte colony-stimulating factor (G-CSF), interferon gamma (IFN-γ), IFN-γ-inducible protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein-1β (MIP-1β), and vascular endothelial growth factor (VEGF). When plasma and passive drool saliva samples were compared, only 3 cytokines, IL-6, IFN-γ, and MIP-1β, were statistically significantly correlated 49 . Glutathione (reduced glutathione [GSH] and oxidized glutathione [GSSG]) is an anti-oxidant that responds to both xenobiotic and endogenous compounds. Ngamchuea et al. 60 found a weak correlation between salivary and whole blood glutathione content (GSH+2GSSG) in healthy participants. Almadori et al. 26 suggested that salivary glutathione levels may be an index of oxidative stress at the level of the upper airways and in particular of the oral cavity and pharynx. A study by Sharma et al. 61 showed a significant and gradual increase in serum and salivary L-fucose between control and oral potentially malignant disorders (OPMDs) or oral cancer (OC) samples. The authors suggested that L-fucose can be used as a reliable biomarker and saliva can be used as a diagnostic fluid for the screening and early detection of OC. A significant positive correlation was found between serum and salivary Cyfra 21-1, serum Cyfra 21-1, and CK19 mRNA expression and between salivary Cyfra 21-1 and CK19 mRNA expression 62 . The magnesium concentration was low in both the blood plasma and saliva of OSCC when individuals with potentially malignant disorders were compared to healthy subjects. Thus, the magnesium ion concentration in blood plasma and saliva could be considered as a tumor marker, playing an important role in carcinogenesis 63 . Oral pre-malignancy and malignancy patients were reported to have lower serum albumin levels compared to healthy individuals. Otherwise, salivary albumin levels were found to increase in oral pre-malignancy and oral malignancy cases compared to healthy individuals. A study by Metgud and Patel 64 suggested that albumin may play a role in early diagnosis and prognosis of oral premalignant and malignant tissues.
Salivary biomarkers are a very promising noninvasive approach to oral cancer detection. In monitoring the disease progression and patient’s response to therapeutics, salivary biomarkers show many advantages because of the noninvasive and cost-effective sampling methods. However, the current challenges in salivary biomarker research are standardizing sample collection, improving sample processing and storage, and reduce the wide variability in cancerous and non-cancerous individuals. Useful biomarkers in the screening and early diagnosis of OSCC are still under research but will be defined in the near future.
Candidates for salivary biomarkers in oral squamous cell carcinoma based on carcinogenesis-related factors
|Angiogenesis-related markers||Inflammation-related markers||Metastasis-related markers||Oxidase stress-related markers||Metabolism-related marker|
||-8-OHdG DNA damage marker (8-OHdG)
||-Non-organic compound: Na, Ca, F, Mg
(VEGF: vascular endothelial growth factor, IL: interleukin)
Description of oral disease proteomic analysis using unstimulated whole saliva (USWS)20
|Disease||Type of saliva||Proteomic approach||Proteins identified||References|
|Oral squamous cell carcinoma||USWS||Mass spectrometry (MS) and western blotting||Increased abundance of myosin and actin.||de Jong et al. 21|
|USWS||Using shotgun proteomics approach (RP-HPLC, CP-LC with TOF and immunoassay)||Detection of 52 protein that presented in diseased samples but absent in healthy samples||Hu et al. 25|
|USWS||Using ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) with hydrophilic interaction chromatography mode||↑Level of choline, betaine and pipecolinic acid
||Wang et al.22|
|Oral leukoplakia||USWS||Two-dimensional gel electrophoresis, mass spectrometry, immunohistochemistry||22 spots very abundant among them apolipoprotein A1, alpha-amylase, cystatins, keratin 10, lysozyme precursor, and CK10 were relevant to the study.||Camisasca et al. 23|
|Proliferative verrucous leukoplakia||USWS||Mass spectrometry||Angiotensinogen (AGT) and dipeptidyl peptidase 1 (DPP1)||Flores et al. 24|
|Premalignant lesions||USWS||Western blotting, mass spectrometry||Salivary actin and myosin||de Jong et al. 21|
Protein biomarkers in USWS for the detection of OSSC20
|Candidate biomarkers||Techniques||Clinical significance||References|
|Interleukin (IL)-6, IL-8, IL-1α, IL-1β, TNF-α, tissue polypeptide antigen (TPA), Cyfra 21-1, cancer antigen 125 (CA 125), telomerase, Mac-2 binding protein (M2BP)||ELISA||Proinflammatory and proangiogenic cytokines found to be indicators of carcinogenic transformation from oral precancerous lesions to oral cancer.
||Katakura et al. 28
|CD44, CD59, Profilin, MRP14||Immunoblot||CD44 and CD59 are the very high sensitive cancer and benign diseases differentiate markers.
||Hu et al. 25
|Glutathione||HPLC||Epidemiological marker for chemoprevention identifies the risk of development of OSCC.||Almadori et al. 26|
|Mac-2 binding protein (M2BP), Squamous cell carcinoma antigen 2, involucrin, calcyclin, cathepsin-G, azurocidin, transaldolase, carbonic anhydrase I, calgizzarin, myeloblastin, vitamin D-binding protein||ELISA, shotgun proteomics||M2BP is for detection of OSCC this biomarker gives a sensitivity of 90% and a specificity of 83%, and all of them serve as a clinical tool for the noninvasive diagnosis of OSCC.||Hu et al. 25|
|Immunoglobulin heavy chain constant region gamma (IgG), S100 calciumbinding protein, cofilin-1, transferrin, fibrin||LC/MS||IgG known to be an inhibitor of apoptosis, S100A2, an 11.4 kDa protein that is a prognostic biomarker for OSCC, cofilin proteins are involved in cancer progression, metastasis, and angiogenesis. Transferrin levels in saliva are associated with the size and stage of cancer. Fibrin in OSCC is involved in several carcinogenic processes.||Jou et al. 27
|α-1-antitrypsin (AAT)||2DE||AAT is useful for the prediction and determining the aggression of OSCC.||Righini et al. 34|
|Secretory leukocyte peptidase inhibitor (SLPI), cystatin A, keratin 36, thioredoxin, haptoglobin (HAP), salivary zinc finger, protein 510 peptide, a-amylase, and albumin||MS-based proteomics||SLPI, cystatin A, keratin 36 are potentially involved in the preventive treatment of OSCC. Thioredoxin mRNA levels are elevated in oral cancers and in other cancers as well. Salivary zinc finger, protein 510 peptide, a-amylase, and albumin are useful in the early detection of OSCC.||Reddy et al. 35
Transcriptomic biomarkers identified in USWS for OSSC detection20
|Candidate biomarkers||Techniques||Clinical significance||References|
|Interleukin (IL)-1 , IL-8||ELISA||Angiogenesis, cell adhesion, chemotaxis, immune response, replication, signal transduction, proliferation, inflammation, and apoptosis||Li et al. 37
|Dual specificity phosphatase 1 (DUSP1)||Quantitative PCR (qPCR) and microarrays followed by qPCR||Oxidative stress, protein modification, signal transduction||Li et al. 37|
|H3 histone family 3A (H3F3A)||qPCR and microarrays followed by qPCR||DNA binding activity||Li et al. 37|
|Long noncoding HOTAIR||qPCR and microarrays followed by qPCR||Expression of HOTAIR is associated with p53 gene and causes DNA damage||Tang et al. 39|
|miR-125a, miR-200a, miR-31||qPCR and microarrays followed by qPCR||Posttranscriptional regulation by RNA silencing complex, cellular growth, and elevated levels in proliferation in OSCC||Liu et al. 40
(USWS: unstimulated whole saliva, OSCC: oral squamous cell carcinoma, ELISA: enzyme-linked immunosorbent assay, PCR: polymerase chain reaction)
Metabolomics biomarkers identified in USWS for OSCC detection20
|Candidate biomarkers||Techniques||Clinical significance||References|
|Cadaverine, alanine, serine, glutamine, piperidine, taurine piperidine, choline, pyrroline hydroxycarboxylic acid, beta-alanine, alpha-aminobutyric acid betaine, tyrosine, leucine+isoleucine, histidine, tryptophan, glutamic acid, threonine, carnitine, pipercolic acid, lactic acid, phenylalanine and valine||Capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS) and HPLC with quadrupole/TOF MS||Facilitates the clinical detection of OSCC and improves its diagnosis and prognosis. They have a high level of predictive value and serves as a stratification tool.||Wei et al. 42
|Hypoxanthine, guanine, guanosine, trimethylamine N-oxide, spermidine, pipecolate, methionine||CE-TOF-MS||Discrimination of controls from OSCC patients and all of these metabolites have elevated levels in saliva, and hence can be used in noninvasive oral cancer screening.||Ishikawa et al. 44|
(USWS: unstimulated whole saliva, OSCC: oral squamous cell carcinoma)
Commercially available saliva collection devices and their key advantages20
|Device name||Company (city, country)||Dose/volume||Patent No.||Characteristics|
|OraSure||OraSure Tech. (Bethlehem, PA, USA)||The pad draws oral fluid, rich in antibodies||US8062908B2||U.S. Food and Drug Administration (FDA) approved for HIV-1 testing. Easy and safe for public health screening, life insurance risk assessment, and good for outreach community programs.|
|Quantisal||Immunalysis Co. (Pomona, LA, USA)||1 mL±10%||US8641642B2||Contains cellulosic (paper-based) absorbent material for collection of saliva.
|Salivette||Sarstedt AG & Co. KG (Nümbrecht, Germany)||1.1±0.3 mL||US9113850B2||Wide application range, including detection of HIV antibodies, oxidative stress steroid hormones for general wellness.
|UltraSal-2||Oasis Diagnostics Co. (Vancouver, WA, USA)||Up to 24 mL of the whole saliva||US9113850B2||Large amount of saliva (24 mL).
|Greiner Bio-One SCS||Greiner Bio-one (Monroe, NC, USA)||4 mL of a tartrazine||US 20090017442||Only device with internal dye (tartrazine) for 2 minutes used as a saliva quantification tool. Uses a colorimetric method.|
|RNAPro•SAL||Oasis Diagnostics Co. (Vancouver, WA, USA)||1.0 mL of saliva in 1-3 min||US, EU
||Simultaneous collection of RNA and proteins, including cell-free DNA, cell-free RNA, and exosomes. Large DNA and interfering factors removed. Useful for exploration of the salivary transcriptome and the salivary proteome. Built-in Sample Volume Adequacy Indicator (SVAI).|
|Pure•SAL||Oasis Diagnostics Co. (Vancouver, WA, USA)||4.0 mL||US, EU
||Collection of cell-free DNA, cell RNA total RNA, or proteins. Major impurities removed by a built-in filtration system. Built-in SVAI.|
|Super•SAL||Oasis Diagnostics Co. (Vancouver, WA, USA)||1.0 mL||US, EU
||Whole saliva collection system. Absorbent pad material removes interfering mucinous material. Shorter collection time due to the higher surface area of pad material exposed to fluid in the oral cavity.|
|Versi•SAL||Oasis Diagnostics Co. (Vancouver, WA, USA)||A maximum sample volume of 1.4 mL||Patents pending||Whole saliva collection system. Absorbent pad material removes interfering mucinous material.
|Pedia•SAL||Oasis Diagnostics Co. (Vancouver, WA, USA)||A passive collection process with soother design to not only collect but also relax the infant.||Patents pending||Device for passive saliva collection from neonates and infants. Based on pacifier design. Collect whole saliva.|
|DNA•SAL||Oasis Diagnostics Co. (Vancouver, WA, USA)||3 mL||US D627882||Saliva DNA Collection Kit – uses a buccal cell scrape followed by an oral rinse.|
|SimplOFy||Oasis Diagnostics Co., Vancouver, WA, USA||2.0 mL of whole saliva||US20180235206;
||Saliva DNA Collection Kit for genomic DNA – consumer-oriented device, collects whole saliva by expectoration (spitting).|
|Micro•SAL||Oasis Diagnostics Co. (Vancouver, WA, USA)||0.5 mL||US and EU Patents including US
||Device for collection of saliva from infants and neonates. Separate configuration available for collection from small animals.|
|OraGene Dx||DNA Genotek (Ottawa, ON, Canada)||2 mL||7,482,116; 8,221,381; D631,554 S; D640,795 S; 9,079,181; 9,523,115||Saliva DNA collection kit for consumer-based genomic DNA collection.
|OraGene Discover||DNA Genotek (Ottawa, ON, Canada)||1 mL||OGR-500, OGR-500.005 kits: D640,795 S; 9,079,181; 9,523,115||Saliva DNA collection device for research applications|
|OraGene RNA||DNA Genotek (Ottawa, ON, Canada)||2 mL||8,221,381; 9,207,164; 10,000,795||Saliva RNA collection device|
|ORAcollect||DNA Genotek (Ottawa, ON, Canada)||1 mL||7,482,116; 8,728,414; D631,350 S and patent pending||Swab-based device for oral DNA collection. Pediatric version available as a separate product|
|ORACOL||Malvern Medical Developments (Worcester, UK)||1 mL whole saliva||US8641642B2||Foam/sponge device on a stick, mainly used for infectious disease antibody testing particularly measles, HIV, hepatitis A and B, mumps, syphilis, and rubella, but has also been used for substance abuse testing.|
|SalivaBio||SalivaBio LLC (State College, PA, USA)||2 mL||EP2745112A1||Range of products for collecting whole saliva from adults, children, and neonates.|
|i-Swab||Mawi DNA Tech. (Hayward, CA, USA)||1 mL||US20140243706A1||Collection of salivary DNA using swab-based materials|
|Saliva DNA Collection Device||Norgen Biotek Co. (Thorold, ON, Canada)||2 mL||Patents pending||Expectoration (spitting) technique.|
Candidates for salivary biomarkers in oral cancer: based on biomolecules
|Genomic markers||Salivary transcriptome markers (mRNA)||Salivary protein markers||Other markers|
|-Mutation p53 genes codon 63
||-Presence of HPV and EBV