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Salivary epigenetic biomarkers in head and neck squamous cell carcinomas

The early detection of head and neck squamous cell carcinoma (HNSCC) continues to be a challenge to the clinician. Saliva as a diagnostic medium carries significant advantages including its close proximity to the region of interest, ease of collection and noninvasive nature. While the identification of biomarkers continues to carry significant diagnostic and prognostic utility in HNSCC, epigenetic alterations present a novel opportunity to serve this purpose. With the developments of novel and innovative technologies, epigenetic alterations are now emerging as attractive candidates in HNSCC. As such, this review will focus on two commonly aberrant epigenetic alterations: DNA methylation and microRNA expression in HNSCC and their potential clinical utility. Identification and validation of these salivary epigenetic biomarkers would not only enable early diagnosis but will also facilitate in the clinical management.

Yenkai Lim1, Charles Xiaohang Sun1,2, Peter Tran1,2 & Chamindie Punyadeera*,1 1 The School of Biomedical Sciences, Institute of Health & Biomedical Innovations, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, Brisbane, QLD 4001, Australia 2 School of Dentistry, The University of Queensland, 288 Herston Rd, Herston, Brisbane, QLD 4006, Australia *Author for correspondence: Tel.: +61 7 3346 3891 Chamindie.punyaadeera@ qut.edu.au

First draft submitted: 29 August 2015; Accepted for publication: 8 January 2015; Published online: 18 February 2016 Keywords:  DNA methylation • epigenetic biomarkers • head and neck squamous cell ­carcinoma • microRNA • salivary diagnostics

Head and neck squamous cell carcinomas (HNSCCs) is a broad term used to describe malignancies arising from the nasal cavity, paranasal sinuses, oral cavity, salivary glands, pharynx and larynx [1,2] . A large number (>90%) of these malignancies are predominantly of the squamous cell carcinoma (SCC) type that arise predominantly from the squamous epithelium of the oral cavity and the oropharynx  [2–5] . HNSCC currently stands as the sixth most common cancer worldwide with 900,000 new cases and 300,000 deaths annually  [1] . Patients with HNSCC have a 5-year mortality rate of 40–50% due to the lack of effective screening techniques and early diagnostic methods [3,6,7] . In addition, the low survival rate is often associated with late diagnosis of the disease [8] . A survey carried out in the USA demonstrated that greater than 30% of HNSCC cases were diagnosed at an early stage (stage I and stage II) while

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greater than 60% cases were diagnosed at an advanced stage (stage III and IV), highlighting the importance of early diagnostic ­methods  [8–10] . The risk factors for HNSCC include smoking, alcohol consumption, betel nut chewing, poor oral hygiene and genetic predisposition  [1,2,11–13] . Epstein–Barr virus infection as well as eating cured meat seems to be associated with the development of nasopharyngeal SCC (one of the subtypes of HNSCC) [2] . While the prevalence of smoking and alcohol consumption has continued to decline over the past decade, the global incidence of HNSCC is on the rise and this has been attributed to an increase in human papillomavirus (HPV) infection  [2,14] . HPV-negative HNSCC presents more commonly in males (2:1) and carries a significant predilection in people of 40 years and above. However, an emerging subgroup

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Review  Lim, Sun, Tran & Punyadeera of HPV-positive HNSCC presenting in young people have highlighted the impact of additional lifestyle risk factors such as having oral sex and/or sex with same gender and/or multiple sexual partners [2] . The SEER database has documented a 225% increase in HPVpositive HNSCC from 1988 to 2004 in contrast to a 50% decline in HPV-negative HNSCC [15] . More importantly, both of these cancer types (HPV-negative and HPV-positive) have diverse biological and clinical features that require targeted clinical management due to their differential behavior and responses to chemoradiotherapy  [16] . This review will elaborate on the potential utility of salivary epigenetics biomarkers and their implications on clinical decision making in HNSCC. Human saliva as a diagnostic medium to detect head & neck squamous cell carcinoma Saliva is an important biofluid that is often overlooked as a diagnostic medium. Saliva is produced by three paired major salivary glands and numerous minor salivary glands that excrete saliva into the oral cavity at a flow rate of 1–1.5 l/day [17] . The major salivary glands include: the parotid glands (located next to masseter muscles and opposite to maxillary first molars), submandibular glands (located in the floor of the mouth) and sublingual glands (located in the floor of the mouth) are anatomically large in size in comparison to small minor salivary glands which can be found throughout the oral cavity in locations such as lower lip, tongue and pharynx [17] . Histologically, the functional units of salivary glands are mucous and serous acini cells that produce and determine the type of saliva secreted. Serous acini cells produce secretions enriched with proteins and water while mucous acini cells secrete ‘slippery fluid’ enriched with glycoproteins and water [17] . Saliva is then secreted through a series of ductal structures that modulate the electrolytic composition ultimately leading to its secretion and mixing in the oral cavity as referred to by whole saliva [17] . The variable origins of saliva excreted into the oral cavity carry a potential confounding issue in salivary diagnostics and it is important to acknowledge this in the study of ­salivary biomarkers. The composition of whole saliva includes a large number of inorganic materials such as calcium, phosphate and bicarbonate as well as organic material such as mucin and immunologlobins [17] . Together, they serve to assist important biological, chemical and physiological functions such as lubrication for speech, buffering acidity, facilitating remineralization to dental tissue, antibacterial actions, taste and digestion [17] . Saliva quantity and quality is affected by factors such

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as systemic diseases including autoimmune conditions, pharmacology and irradiation and subsequently these factors must be carefully accounted for when viewing saliva as a diagnostic media [18] . In general, healthy individual’s saliva is at a pH of 7.4, which is slightly basic and facilitates the optimal preservation of intact high-molecular weight DNA. As such, DNA derived from salivary cells are considered as an optimal DNA source not only for disease detection but also for disease monitoring [14] . As such, saliva may be useful in epigenetic studies. Saliva is emerging as an alternative diagnostic medium to detect both local and systemic events. While tumors within the oral cavity may shed cellular material directly into saliva; oropharynx tumors may shed tumor cells into blood that get transported into saliva through processes of diffusion, active transport and ultrafiltration [14,19] . This allows saliva to be considered as a ‘sampling matrix’ for many molecular and physiological processes and may reflect aberrant pathological changes that occur in distant organ systems  [14,19] . This enabled researchers to utilize saliva as a sampling to investigate novel diagnostic biomarkers in saliva for distant cancers such as the breast and stomach cancers [20,21] . In addition to biomolecular sampling, cellular sampling can also be achieved using saliva. Traditionally, obtaining cellular samples in occult anatomical regions such as oropharynx and tonsillar crypts were considered challenging and required specialized procedures such as a cytobrush or needle biopsies. However, cells from these regions are constantly exfoliated into the oropharyngeal environment which could potentially be sampled by using simple oral rinses and gargles, providing an increasingly popular and simple solution to current clinical ­challenge  [14,22] . Over the past decade, saliva as a diagnostic medium has gained increasing attention especially in the field of HNSCC detection. Saliva as a diagnostic medium carries a number of advantages compared with conventional diagnostic media, such as blood, urine and tissue testing. First, saliva is produced and collected locally near the sites of oral and oropharyngeal malignancies and by analyzing saliva we are able to detect and predict tumor activities. The close proximity of the diagnostic medium to the site of the primary tumor enables sampling tumor-specific biomolecules by analyzing saliva with minimal interference. Saliva is able to sample otherwise occult regions of the tonsillar crypts which have traditionally been a diagnostic challenge for visual and physical examination [14] . Second, saliva sampling is minimally invasive and easy to perform. The noninvasive nature of saliva collection facilitates easier patient management in challenging clinical

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Salivary epigenetic biomarkers in head & neck squamous cell carcinomas 

Review

Table 1. Commercial saliva collection devices that can be tailored in epigenetic studies. Saliva collection device(s)

Target biomolecules

Information

Salimetrics oral swabs

DNA, protein

www.salimetrics.com

DNA.SALTM; Mini.SALTM and Midi.SALTM

DNA

www.4saliva.com

RNAPro.SALTM

RNA

 

Pedia.SALTM (for pre-term infant use)

DNA, RNA, protein

 

Greiner Bio-One Saliva Collection System

DNA

www.gbo.com

OraGene DNA

DNA

www.dnagenotek.com

OraGene RNA

RNA

 

Saliva Collection, Preservation and Isolation Kit

DNA

https://norgenbiotek.com

situations such as dealing with small children, noncompliant and anxious patients [19] . Furthermore, the readily available nature of saliva collection also permits multiple samples to be collected from an individual for validation and follow-up [14] . The latter makes saliva as an attractive fluid for home testing. Even though saliva sample collection is easy and straightforward, saliva sampling is subjected to multiple confounding variables, in other words, method of sampling (unstimulation vs stimulated saliva), flow rates, age, drug use, sex, diet, physiological status and salivary gland pathology [19,23] . All of the above factors may affect the concentrations of analytes present in saliva. There is also the lack of knowledge about saliva physiology and biomolecular transportation to and from saliva. Some biomolecules in saliva are affected by the diurnal and circadian rhythms and this makes it challenging to use saliva as a diagnostic medium [19] . As an example, salivary growth hormone levels are elevated in a morning saliva sample as compared with an afternoon collection and as such this should be taken into consideration when using growth hormone levels in diagnosing disease [19,24] . Furthermore, analytes present in saliva are 100–1000-fold less concentrated than those present in blood and therefore requires highly sensitive detection technologies to translate salivary diagnostic assays from bench to the bedside of a patient [19] . Rantonen et al. found a positive correlation between salivary and serum growth hormone levels, albeit salivary growth hormone concentrations were 1000-fold lower than serum levels [24] . Recent advances in saliva diagnostic technologies have provided some promising high sensitivity assays that can detect low amounts of proteins present in saliva. As an example, Punyadeera et al. documented significantly higher C-reactive protein levels (1680 vs 286 pg/ml) in the saliva collected from ischemic heart disease patients compared with healthy controls using AlphaLISA® technology [25] . Furthermore, Wang et al. demonstrated the clinical utility of using cell-free tumor

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DNA in saliva as a biomarker to diagnose oral cavity squamous cell carcinoma (100% detection rate was observed in saliva vs 80% detection rate in serum) [26] . In contrast, serum was found to be the preferred medium for other anatomical sites besides oral cavity (oropharynx, larynx and hypopharynx) squamous cell carcinoma (saliva detection rates 47–70% vs 86–100% detection rates in serum) [26] . The most common saliva sampling methods include unstimulated whole mouth saliva collection, stimulated saliva collection and oral rinse method [14,19] . Unstimulated whole mouth saliva collection is simple, easy and represents saliva produced by three major salivary glands, minor salivary glands as well as gingival crevicular fluid (GCF) secretions, and is commonly done by draining/drooling method [14,19,27,28] . In contrast, stimulated saliva collection is done by chewing onto a paraffin wax (mechanical stimulation) or application of small amount of 0.1–0.2 mol/l citric acid (acid stimulation) [19] . For patients with compromised salivary flow rates as a result of impaired salivary gland functions (i.e., aging, medication, chemo/radiation therapy), stimulated saliva may be a more convenient sampling option for diagnostic purposes [29,30] . Oral rinse sampling, on the other hand is excellent for ‘hardto-reach’ areas within the head and neck region such as the oropharynx, hypopharynx and larynx. This is especially helpful in HPV-positive HNSCC studies as HPV–16 commonly reside in the tonsillar crypts within the oropharynx [22,31–33] . Apart from the aforementioned conventional methods, new saliva collection devices are emerging in the market (Table 1) and have gained increasing popularity in clinical practice due to their improve ease of use, cost-effectiveness and minimal patient discomfort [14,19] . Mohamed  et al. observed that salivary collection devices as well as salivary flow rates have an impact on protein detection [34] . They found that the processing methods had an adverse effect on the concentration of the total protein detected as well as on individual

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Review  Lim, Sun, Tran & Punyadeera protein concentrations [34] . Similarly, Topkas et al. demonstrated significant differences in protein levels (C-reactive protein, myoglobin and immunoglobin E) based on the collection method used [23] . Morzel et al. also found that the saliva proteome is substantially altered in infants as their diets transitioned significantly in a time span of 3 months [35] . These findings suggest potential interactions between salivary proteins with oral physiology and diet [23,35] . While salivary diagnostics remains an exciting avenue for biomarker development, it is important to acknowledge the limitations with the use of saliva. As such, early results should be interpreted with caution in conjunction with consideration for clinical findings in order to achieve an optimal outcome. Biomarker development pathway Due to the clinical relevance of biomarkers in clinical decision making, it is vital to understand the biomarker development trajectory, which follows a well-defined systematic approach. Numerous authors have proposed stepwise development pathway (Figure 1) [36,37] . Phase I: identification

The identification phase involves the preliminary establishment of the potential biomarker candidates. The selection of the candidates may take place by two different approaches. The ‘knowledge-based’ or ‘targetdriven’ approach, indicates that the selection is derived from existing knowledge or scientific deduction, and only involves a limited number of biomarkers [36] . The second approach is referred to as the ‘shotgun’ or ‘unbiased’ approach and involves the examination of Biomarker candidate

Biomarker candidate

Biomarker candidate

Biomarker candidate

Phase I

Phase II

Validation and Phase IV translation

ma

Verification and optimization

rke r pi

Assessment

Bio

Phase III

pel

ine

Identification

Clinical biomarker

Figure 1. Biomarker development pathway.

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numerous biomolecules either in tissues or body fluids (e.g., certain chemical solvents, genetic molecules or proteins) and covers many candidates [36,37] . The latter approach may lead to novel findings, however this process is relatively time intensive and financially demanding [37] . Phase II: assessment

Following the identification of candidate biomarkers, the primary suitability of the candidates is assessed. Comparison between normal and diseased samples is performed to ensure that there is a significant difference in abundance between the normal and diseased statuses [37] . Comparison may occur either in a binary (positive/negative) or ordinal (numeric value) fashion. If binary comparison is used, a preliminary value of true positive rate (TPR, or sensitivity, which is the proportion of case subjects that are positive for a certain biomarker) and false positive rate (FPR, or 1-specificity, which is the proportion of control subjects that are biomarker-positive) will be recorded [36] . If a quantitative (ordinal) comparison is performed and a threshold level is not performed, a receiver operating characteristic (ROC) curve is used to indicate the various TPR and FPR at various threshold levels [36] . During this step, a reliable assay is established to evaluate and standardize the candidate biomarker suitability [36] . During both Phase I and Phase II, the focus is to confirm the association between biomarker and the disease status  [37] . The significance of sensitivity and specificity depends on the application. [37] . Phase III: verification & optimization

During the verification and optimization phase, potential candidate biomarkers that are deemed suitable and have previously undergone robust evaluation would undergo verification within a large clinical study [36] . Multi-biomarker panel maybe developed depending on the optimization of the algorithm [36] . Longitudinal comparisons are desirable in order to compare withinsubject variability and may reveal a different sequential testing biomarker scheme [36] . Candidate biomarkers are further optimized, in terms of their sensitivity, specificity and potential for clinical application or commercialization. Phase IV: validation & translation

If a candidate biomarker performance is satisfactory and meets the clinical sensitivity and specificity for a desired application, the potential candidates can then move toward the final phase of clinical validation and translation  [37] . Large-scale clinical validation trials of the candidate biomarkers are performed [37] . The performance of the candidate is tested in a more general-

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Salivary epigenetic biomarkers in head & neck squamous cell carcinomas 

ized population and its clinical sensitivity, specificity, PPV and NPV may undergo further modifications [36] . The biomarker validity is ultimately assessed in a diagnostic environment and its ability to detect target disease determines its translational potential [36] . A successful biomarker may undergo further scrutinization depending on the long-term reduction of the disease mortality either within the general population and/ or at risk groups [36,37] . The final step of this pathway usually takes tremendous amount of time and financial investment and unfortunately only a small percentage of the candidate biomarkers may progress toward a clinical assay [36,37] . Owing to a lack of timely diagnosis and effective screening regiments, HNSCC is often diagnosed only when patients present with symptoms such as limitations of tongue mobility, difficulty breathing, generalized orofacial pain, and difficulty in mouth opening. Unfortunately, these symptoms are secondary to growth of a primary tumor or critical anatomical involvement that signifies a late clinical staging where prognosis is most poor and intervention outcomes most morbid. With the exception of oral cavity where significant literature has documented the existence of clinically monitorable potentially malignant disorders such as leukoplakia or erythroleuplakia, the existence of a potentially malignant disorder of the oropharynx have not been comprehensively described [38,39] . As such, an unmet clinical demand is evident to supplement visual and tactile screening performed by health practitioners. As such salivary biomarkers may eventuate within a clinical setting facilitating opportunistic screening service that may be deliverable by a broader range of people due to decreased technique sensitivity. Biomarkers need to be measurable, specific, sensitive, predictable, and robust indicators for a particular clinical application that can have a positive impact on clinical utility and decision making. Reflecting the clinical demand, HNSCC biomarkers can be extrapolated from the clinical disease and management model as represented in Figure 2. Namely, HNSCC biomarkers can be developed to have the potential clinical application for population screening, diagnostic, ­prognostic, or predictive purposes. The application of biomarkers for population-based screening of HNSCC must account for its relatively low incidence within a population. A large number of individuals will be without the disease and as such, biomarker assays must have adequate NPV in addition to low cost requirements and ease of biomarker sampling and evaluation. Conversely, biomarkers employed in diagnostics should display properties such as high clinical sensitivity, specificity and commercial potential and is also ideal to be integrated into a low

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Review

cost point-of-care testing platform. Moreover, biomarkers that are being utilized for predictive purposes in monitoring tumor recurrence require known reference cut-off values to dictate clinical decision-making. Subsequently, the desirable properties of biomarkers vary with their intended clinical performance. Many types of biomarkers have been explored in HNSCC ranging from genomics, proteomics, metabolomics and methylomics. Additionally, many noninvasive biological sources (cell scrapings, blood, saliva and urine) have also been explored [40] . Epigenetics Epigenetics changes have been described as alterations to the DNA double helix structure without changing the underlying genetic sequences, causing phenotypic or gene expression changes within a cell [41,42] . Epigenetics mechanisms include DNA methylation, DNA hydroxymethylation, microRNA (miRNA), long noncoding RNA (lncRNA) and histone modifications associated with gene silencing and or activations [41– 45] . Alterations within the confinement of any of these mechanisms will result in irregular gene expression, leading to epigenetically regulated diseases such as cancers  [41–48] . Epigenetic events generally occur in the initial phase of the carcinogenesis and as such provide valuable information into the disease pathology  [49] . Besides the potential clinical utility of epigenetic diagnostics, epigenetic changes have also been implicated in cancer progression albeit this research is in infancy [50–52] . These findings hold the potential of offering better diagnosis and/or prognosis of cancer leading to personalized medicine. As the field of epigenetics advances, new linkages between environmental-induced epigenetics modifications with diseases are emerging [42] . As an example, diet, ultraviolet exposure and tobacco consumption are known to catalyze DNA methyltransferases (DNMTs), ultimately leading to tumorigenesis [53–55] . DNA methylation is the most common and well known cause of epigenetic modification in mammalian cells [41,45] . It is essential for embryonic development, cell-cycle regulation and cell differentiation [45] . DNA methylation is regulated by a number of DNMTs, an enzyme that induces nucleophilic attack on the cytosine residue at the sixth carbon [47,56] . This attack increases the negative charge on the fifth carbon and as a consequence, attracts and binds nearby methyl-group bound to AdoMet  [47,56] . The newly added methyl-group forms an extension on the overall DNA alpha-helical structure, interfering with the binding of transcription factors to the promoter region to initiate transcription  [57] . DNA methylation most commonly occurs among CpG islands (cytosine residue followed by a

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Prognosis Etiology

Pathogenesis Screening

Management or treatment

Disease Diagnosis

Prediction

Figure 2. The clinical disease model and concurrent demand for novel biomarkers.

guanine residue), and is commonly found at the promoter region of tumor suppressor genes [41,42,45] . DNA methylation in promoter regions of tumor suppressor genes blocks the transcription of these genes, potentially resulting in tumorigenesis [4,57,58] . DNA methylation can also occur away from the promoter regions of tumor suppressor genes and potentially causes the initiation of tumorigenesis [59] . As an example, the addition of methyl group will alter the UV absorption spectrum for cytosine to a region that is significantly more receptive to sunlight. This alteration predisposes the DNA to spontaneous deamination, UV-induced mutation and hydrocarbon carcinogenesis [59] . Therefore, epigenetic biomarkers may aid clinicians to detect the disease at an early stage, stratify risk, monitor ­disease progression and indicate timely intervention [42] . The advancement of high-resolution microarray and genome-wide sequencing technologies has identified miRNAs and lncRNA as crucial regulatory molecules involved at the transcriptional level [60,61] . miRNAs are small noncoding RNAs (18–22 nt), previously referred to as ‘junk DNA’ and are established regulators of cellular development, cell identity and cellular physiology [28,62] . In contrast, lncRNA are >200 nt and share many common features with mRNA [60,61] . There are approximately 2000 known human miRNAs and new candidates are continuously being discovered. miRNAs regulate more than 60% of mammalian cellular gene expression by binding to mRNA [62,63] . This process inhibits mRNA translation and/or facilitates the degradation of the targeted mRNA [44,48] . miRNAs are usually found within the introns or in the intergenic regions  [48] . DNA polymerase II is responsible for the transcription of most miRNAs [48] . Since 50% of the human promoters are rich in CpG islands, DNA methylation could potentially serve as one of the epigenetics mechanism for silencing miRNA expression in various tissue types [48] . Over the past decade, several miRNAs were found to be downregulated in over 20 different types of cancers including breast, cervix, head and neck, lung, prostate, stomach and bladder [44,64] . DNA hydroxymethylation is an intermediate product of the demethylation due to oxidation reactions  [65–67] . The level of DNA hydroxymethylation often decreases during differentiation, suggesting that it may be associated with cellular differentia-

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tion  [65–67] . Histone modification occurs predominantly within the histone amino terminal tails protruding from the surface of the nucleosome as well as on the globular core region [42,43,46] . These modifications include lysine acetylation; lysine and arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and sumoylation or in combination to form the histone code [41–43,45,46,48] . The functionality of histone modification is to hide DNA away from nearby transcription factors to prevent gene expression [42] . Disruption of the histone code risks the exposure of DNA and promotes transcription, leading to tumorigenesis [42] . Although, the function of DNA hydroxymethylation is not fully understood, a decrease in the global hydroxymenthylation has been reported in several type of cancers [65–67] . Histone modifications are frequently altered in tumor cells and this alteration while not fully elucidated may warrant future investigation [44,46] . However, this alteration causes overexpression of targeted epigenetic regulators, leading to the silencing of involved miRNAs [44] . Consequently, the activity of epigenetic regulators could also be distorted by oncogenes or tumor suppressor genes in tumor cells, resulting in miRNA repression and ­silencing  [44] . Salivary epigenetics biomarkers in head & neck squamous cell carcinoma Numerous studies have been carried out to investigate the association between changes in DNA methyl­ ation and tumorigenesis in HNSCC over the past 5 years [3,7,14,27,68–79] . DNA methylation as an earlier tumorigenicity event is ideal for the early diagnosis of HNSCC  [49] . For example, DNA hypermethylation is common in tumor suppressor genes and as such encourages cancer cells to proliferate continuously, which may eventually result in the formation of neoplasm [14,80] . In addition, major risk factors for the development of HPV-negative HNSCC are alcohol and tobacco consumption. A strong correlation between smoking and tumor suppressor gene promoter DNA methylation in HNSCC is observed [49] . Common tumor suppressor genes in saliva that are hypermethylated in HNSCC include RASSF1α, p16INK4A, DAPK1 and MGMT (Table 2)  [69,72,75,81] . As there is no tumor suppressor gene specific to a certain

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Salivary epigenetic biomarkers in head & neck squamous cell carcinomas 

Review

Table 2. Commonly hypermethylated genes in saliva of head and neck squamous cell carcinoma patients. Gene

Cellular functions

Tumor anatomical site(s)

Type of saliva analyzed

CCNA1

Cell cycle regulator

Oral cavity, larynx, oropharynx, hypopharynx

Oral rinse

DAPK1

Programmed cell death

Oral cavity, larynx, oropharynx, hypopharynx

Oral rinse, unstimulated whole-mouth saliva

DCC

Receptor for nectrin required for axon guidance

Oral cavity

Oral rinse

[3,72,88]

EDNRB

G-protein-coupled receptor

Oral cavity

Oral rinse

[3,72,88,89]

ERCCI

Nuclear hormone receptor

Oral cavity, oropharynx

Oral rinse

[3,90]

HOXA9

Promotes mammary epithelial cell growth and survival

Oral cavity

Oral rinse

[72]

KIF1A

Protein kinase involved in apoptosis and Oral cavity DNA damage response

Oral rinse

[3,72,91]

MED15

RNA polymerase II regulator

Oral cavity

Unstimulated wholemouth saliva

MGMT

DNA mismatch repair system

Larynx, hypopharynx

Oral rinse

[3,85]

MINT31

Calcium channel regulator

Oral cavity, larynx

Oral rinse

[3,78,85]

NID2

Cell adhesion

Oral cavity

Oral rinse

[72]

PAXI

Transcription factor for chordate development

Oral cavity

Oral rinse

[73]

p16INK2A

Receptor of sonic hedgehog

Oral cavity, larynx, hypopharynx Oral rinse, unstimulated whole-mouth saliva

RASSF1α

Induced growth inhibition along the RAS-activated signaling pathway

Oral cavity

Oral rinse, unstimulated whole-mouth saliva

TIMP3

T-cell antigen receptor, recognition of foreign antigens

Oral cavity, larynx, oropharynx, hypopharynx

Oral rinse

cancer type, these biomarkers are often combined together to form an HNSCC-specific biomarker panel. In the case of HPV-positive HNSCC, tumor cells showed global hypomethylation due to the activities of the viral oncoprotein E6 and E7 [82,83] . E6 and E7 are responsible for the inactivation of the tumor suppressor gene products, p53 and Rb, respectively [84] . Global DNA hydroxymethylation is significantly lower in tumor cells compared with normal healthy cells  [92,93] . The two mechanisms accountable for the loss of DNA hydroxymethylation to date are the inactivation due to mutations of TET proteins and IDH 1/2  [92,93] . The inactivation of TET proteins directly hinders oxidative demethylation process while IDH 1/2 enzyme stops the production of (α-KG, which is a co-factor alongside TET [92,93] . Since most cancers do not exhibit mutations in either of these genes, DNA hydroxymethylation may be an ideal epigenetic biomarker for specific cancer types [92,93] . Within the past 5 years of literature, DNA hydroxymethylation was found to be associated with breast and pancreatic cancer [94,95] . A recent study has shown that for the first

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Ref. [3,85,86] [3,14,85,87]

[27]

[3,14,85] [3,14] [3,76,78,85]

time, DNA hydroxymethylation could be detected in saliva samples [92] . However, these results do not correlate with the DNA hydoxymethylation levels found in blood [92] . Histone modifications also play an important role in gene regulation [96] . However, the technique for quantification and evaluation of histone modifications in a noninvasive manner, specifically using saliva has been only established recently by Gasche et al. [96] . In 2012, Kusumoto et al. demonstrated the feasibility of using DNA collected from oral rinse samples to measure the histone modifications of p16INK4A gene using a chromatin immune-precipitation assay in oral squamous cell carcinoma (OSCC) patients [97] . This technique has broadened the application of salivary diagnostic techniques for investigating histone modifications in HNSCC as a potential biomarker [96] . Silencing of multiple miRNAs studied in HNSCC and especially OSCC has been demonstrated and documented in saliva (Table 3)  [28,62,98] . Unlike DNA methylation, global hypomethylation (downregulation) of miRNAs was not observed in HNSCC-posi-

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Table 3. Commonly aberrant miRNA expression changes in saliva of head and neck squamous cell carcinoma patients. miRNA

Expression-levels in saliva of HPV-negative HNSCC patients compared with healthy controls

Saliva collection type

miR–9

Overexpressed

Unstimulated whole-mouth saliva

[28]

miR–24

Overexpressed

Unstimulated whole-mouth saliva

[101]

miR–27b

Overexpressed

Unstimulated whole-mouth saliva

[101]

miR–31

Overexpressed

Unstimulated whole-mouth saliva

[62,102–104]

miR–125a

Underexpressed

Unstimulated whole-mouth saliva

[105]

miR–134

Overexpressed

Unstimulated whole-mouth saliva

[28]

miR–136

Underexpressed

Unstimulated whole-mouth saliva

[101]

miR–137

Overexpressed

Unstimulated whole-mouth saliva

[102]

miR–147

Underexpressed

Unstimulated whole-mouth saliva

[101]

miR–148a

Underexpressed

Unstimulated whole-mouth saliva

[101]

miR–191

Overexpressed

Unstimulated whole-mouth saliva

[28]

miR–200a

Underexpressed

Unstimulated whole-mouth saliva

[105,106]

miR–220a

Underexpressed

Unstimulated whole-mouth saliva

[101]

miR–222

Underexpressed

Unstimulated whole-mouth saliva

[62,103,104]

miR–323–5p

Underexpressed

Unstimulated whole-mouth saliva

[101]

miR–503

Underexpressed

Unstimulated whole-mouth saliva

[101]

miR–632

Underexpressed

Unstimulated whole-mouth saliva

[101]

miR–646

Underexpressed

Unstimulated whole-mouth saliva

[101]

miR–668

Underexpressed

Unstimulated whole-mouth saliva

[101]

miR–877

Underexpressed

Unstimulated whole-mouth saliva

[101]

miR–1250

Underexpressed

Unstimulated whole-mouth saliva

[101]

tive tumor cells, albeit there are several specific miRNAs with different levels of expression in HPV-positive HNSCC [28,62,98] . In the case of lncRNAs, Tang et al. were the first group to investigate salivary lncRNA as a potential biomarker for OSCC [99] . While multiple lncRNAs were differentially expressed between tumors and matched adjacent nontumor tissues, HOTAIR was the only lncRNA quantifiable in saliva at a significant level [100] . As such, salivary lncRNAs may be a p ­ romising avenue of biomarker development in HNSCC. Translation of biomarkers into clinical practice A significant amount of qualitative literature and laboratory case-control studies in genomics, proteomics, metabolomics and methylomics exists on epigenetic biomarkers to HNSCC [107] . However, only a handful of biomarkers have been validated and ­commercialized [108,109] . Critical factors that may hinder translation of biomarkers to the clinic include the need for matched

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Ref.

case and control specimens to prevent incidental biomarker findings due to confounding factors. Classic examples include: not adequately matching case and control subjects by age or gender when investigating biomarkers associated with aging/gender or metabolic disorders. Another significant factor that is overlooked in biomarker development for HNSCC is the use of histologically ‘healthy’ tissue as a control specimen for tumors. These tissues while histologically normal may appear as an attractive alternative due to the perfect matching of cases with controls, may already harbor expression changes that are critical to early malignant transformation. These early changes have been made evident by numerous comparative studies between host-healthy-control tissue and donor-healthy-control tissue studies in prostate cancer, breast cancer and colon cancer [110–112] . Furthermore, changes in tissue specimen are not always translated into other body ­fluids such as blood and or saliva. Guidelines are now emerging and in place to standardize sample collection, processing and analysis based on the PRoBE (prospective-specimen-collection,

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retrospective-blinded-evaluation) guidelines that recommend stringent collection of specimens from participant populations to closely reflect the target group that the biomarker is applied [113] . The current state of biomarker research in HNSCC lends itself to the detection of insufficiently sensitive or specific biomarkers or biomarkers for advanced disease staging that offers little clinical utility, as survival rates are lowest. Subsequently, future studies should attempt to identify novel biomarkers of early lesion transformation in potentially malignant disorders in order to identify those at risk or with occult HNSCC. The use of saliva as a diagnostic medium is not without significant obstacles that must be acknowledged prior to starting research. These include the presence of proteins, inhibitors and enzymes in saliva may alter the abundance and the expression of potential biomarkers during collection, storage and processing, the individual transient variations of biomolecules in saliva secretions as a result of the presence of diurnal and circardian cycles, the interindividual variability of saliva secretions as a result of polypharmacy or systemic disease and variations as a result of analytical techniques employed [34,114] . In order to overcome these potential confounding factors, standardized protocols for sample collection, storage and processing are critical and at best, sample collection and processing should be carried out in a similar fashion as to the intended clinical utility of the biomarker [107] . Future perspective Current diagnosis for HNSCC relies on fine-needle aspiration cytology (FNAC) and/or primary tumor biopsy  [115] . However, some cases of HNSCC start as leukoplakia or erythroplakia lesion(s) in the oral cavity that cannot be constituted to a particular condition or disease [116] . In such cases, neither FNAC nor primary tumor biopsy would comply [116] . Histological examination remains the ‘gold standard’ to determine the prospect of malignancy surrounding the site of leukoplakia and erythroplakia [116] .

Review

While immediate actions such as FNAC and biopsy are taken to access lesions with severe dysplasia, nondysplastic lesions remain to be watched [116] . Studies have shown that 16% of the nondysplastic lesions have the potential to develop into malignant disorder  [117] . While the developments of diagnostic and prognostic biomarkers are fundamental for HNSCC, predictive biomarkers are equally crucial as well and often neglected [118] . HNSCC is the sixth most common cancer worldwide [119] . According to a survey carried out in the UK, early detection of HNSCC is able to reduce the overall in-patient and out-patient expenditures by 14% (half a billion on a global scale) [120,121] . In addition, with early diagnosis patients are also eased from the financial burden and unnecessary treatments. The incidence rate of HPV-negative HNSCC is gradually decreasing in the western world over the past decades due to a reduction in smoking and alcohol consumption [119] . This phenomenon has receded major investors and big pharma companies to develop new drugs for HNSCC as they are mostly situated in the west [119] . In contrast, HPV-positive HNSCC is on the rise especially in the developed world and most patients are young. The development of new drugs often require a large sum that comes with multiple high-level risks [119] . Consequently, HNSCC is considered as a ‘neglected diseases’ as there is no new therapies in the pipeline [119] . Under the circumstances, salivary epigenetics biomarkers are invaluable as they not only allow the early diagnosis of HNSCC but can also stratify patients based on HPV status. Early diagnosis often leads to good prognosis which in turn decreases the mortality rate due to HNSCC [14,80,122] . Salivary epigenetic predictive biomarkers hold tremendous potential when tailoring therapy response in HNSCC patients; when successful, will open up new avenues for targeted therapeutic treatments [118] . Nevertheless, the discoveries of these biomarkers are futile unless the data can be integrated into patient care. To fulfil the utility of discovered biomarkers, it is essen-

Executive summary • Head and neck squamous cell carcinomas are the sixth most common cancer worldwide. • Human saliva may be a viable diagnostic medium for head and neck squamous cell carcinomas due to its local proximity to sites of malignancy and ease and non-invasive nature of collection. • Epigenetic events are implicated in the initial phases of carcinogenesis and provide valuable insights into individual disease progression and personalised medicine. • DNA methylation has been studied as a carcinogenic event and biomarker discovery has revealed hypermethylation of tumour suppressor genes in saliva as potential candidates. • MicroRNAs are dysregulated in head and neck squamous cell carcinoma and saliva microRNAs’ signature could potentially be used in addition to DNA methylation as diagnostic and screening biomarker. • Future research should attempt to identify novel biomarkers to reflect early tumour activities in order to identify those at risk or with occult head and neck squamous cell carcinoma.

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Review  Lim, Sun, Tran & Punyadeera tial for basic researchers and clinicians to work closely together to interpret the laboratory data and translate them into useful clinical information [118] . The mutual respect and understanding shared between basic researchers and clinicians will ultimately lead to better patient clinical management while keeping the ­mortality rate at all-time low [118] . Financial & competing interests disclosure This study was supported by the Queensland Centre for

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