Oral Oncology 48 (2012) 569577

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Oral Oncology
journal homepage: www.elsevier.com/locate/oraloncology

Review

Saliva: A potential media for disease diagnostics and monitoring

Jingyi Liu, Yixiang Duan
Research Center of Analytical Instrumentation, Analytical & Testing Centre and College of Chemistry, Sichuan University, No. 29 Wangjiang Road, Chengdu 610064, PR China

a r t i c l e

i n f o

s u m m a r y
Within the past 10 years, the use of saliva as a diagnostic tool has gained considerable attention and become a well-accepted method. As a diagnostic uid, saliva offers superiority over serum due to both a noninvasive collection method by specially trained persons and a cost-effective approach for screening of large populations. Collection of saliva offers a reduced risk of infection compared to the collection of serum. Moreover, obtaining saliva samples from infant, disabled or anxious patients, is much easier than obtaining other samples. There is a lot of useful components-changing information in saliva when a person is in sick. Therefore, we dene these changing components as biomarkers. The utilization of biomarkers as early predictors for clinical disease not only contributes to the effective prevention and treatment of diseases, but also enhances the assessment of potential health risks. In this article, we have reviewed the properties of saliva, the salivary analysis method for biomarker discovery, and the diagnostic potentials of salivary biomarkers in monitoring and detecting periodontal disease, Oral and Breast cancers, and Sjgrens syndrome. We also discussed some barriers of applications of saliva as a diagnostic media as well as recent improvements. We also prospected the future processing directions of using biomarkers in disease diagnosis and draw a conclusion that saliva is indeed an effective media in various disease monitoring and diagnosis. 2012 Elsevier Ltd. All rights reserved.

Article history: Received 9 September 2011 Received in revised form 28 December 2011 Accepted 26 January 2012 Available online 19 February 2012 Keywords: Saliva Biomarkers Disease diagnosis Oral squamous cell carcinoma Periodontal disease Cancer Sjgrens syndrome

Introduction Early detection of disease plays a signicant role in successful clinical treatment. In most cases of various diseases, early detection and diagnosis lead to a greater survival rate with a reduced chance of the disease re-emerging. Successful monitoring of a disease, especially in its early stage, may also reduce any severe impacts on a patients health or help to prevent and/or delay succeeding complications. The ability to evaluate physiological conditions, trace disease progression, and monitor post-treatment therapeutic resulting through a noninvasive method is one of the primary objectives in the eld of healthcare research. Saliva, a multi-constituent oral uid that can be collected through noninvasive means, has considerable potential for the surveillance of general health and disease. Human saliva contains many kinds of proteins and peptides, each of them carries several signicant biological functions. With the advancement of novel technological means (such as bioinformatics, metabolomics, genomics and proteomics), saliva, as a clinical tool, has become a more and more attractive option because of its ability to mirror both oral and systemic health conditions.1 But in order for saliva-based diagnostics to be useful, two prerequisites must be fullled: (1) discovering biomarkers for
Corresponding author. Tel.: +86 028 85418180; fax: +86 028 85412316.
E-mail address: yduan@scu.edu.cn (Y. Duan). 1368-8375/$ - see front matter 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2012.01.021

various diseases among the complicated composition of saliva, and (2) evaluating the sensitivity and specicity of biomarkers through a series of continuous developments.2 Saliva prole Water is the most abundant component in saliva, representing 99% of salivas total composition. The solid components soluble in the aqueous phase differ from person to person, and can even vary in the same individual at distinct times during a day. The inorganic species are mainly composed of weak and strong ions includ 2+ ing Na+, K+, Cl, Ca2+, HPO2 3 , HCO3 , Mg , and NH3. The organic species (see Table 1) consist of body secretion products (urea, uric acid and creatinine); putrefaction products (putrescine and cadaverine); lipids (cholesterol and fatty acids), and more than 400 types of protein. Among those proteins, the most relevant ones are glandular in origin (alphaamylase, histatins, cystatins, lactoferrins, lysozymes, mucins, and proline-rich proteins (PRPs)) or are plasma-derivatives (albumin, secretory immunoglobulin A (sIgA), and transferrin).3 Human saliva proteome (HSP) analysis is inherently challenging because human saliva contains an inherently large variety of proteins with an equally wide range of concentrations. For example, a-amylase, the most abundant protein in human saliva, is at mg/ ml level, whereas cytokines are typically within the range of pg/ml.4

570 Table 1 Salivary proteins.3 Salivary protein Total proteins a-Amylase Albumin Cystatins group Hystatin Secretory IgA Lactoferrin Lysozyme Mucins group PRPs Statherin Transferrin Origin Functions

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Concentrations 0.47 0.19 mg/ml, 0.9 0.2 mg/ml, 4.3710.0 mg/dl, 2.67 0.54 mg/ml 3257 1682 U/ml, 1080.0 135.6 IU/l, 476 191 lg/ml 0.2 0.1 mg/ml, 0.8192 mg/dl 14.3 kDa form 58 25 lg/ml; 14.2 kDa form 91 46 lg/ml 1190 313 lg/ml 124.3335.3 lg/ml 3.7 2.5 lg/ml 3.592.0 lg/ml, 21.8 2.5 mg/dl, 59.71062.3 lg/ml MUC5B: 2.4 1.7 U/ml Acidic PRP: 456 139 lg/ml, Basic PRP:165 69 lg/ml 4.93 0.61 lmol/l, 36 18 lg/ml 0.58 0.2 mg/dl

Plasma SM > SL P B lymphocytes Mucous > serous SL > SM,P Mucous glands P

Starch digestion Mainly from plasma leakage Antimicrobial(cistein-proteinase inhibitor) Antifungal Antimicrobial Antimicrobial Antimicrobial Lubrication Binding to bacteria and with dietary tannins Ca++ binding

Plasma

SM = submandibular; SL = sublingual; P = parotid.

The reason why saliva can potentially be used as a specimen for diagnosis is because of its exchange with substances existing in human serum. A thin layer of epithelial cells separating the salivary ducts from the systemic circulation enables the transfer of substances to the saliva by means of active carriage, diffusion through the cell membrane, or passive diffusion via a concentration gradient. One of the principal advantages of using saliva as a diagnostic media is that its sampling is easy and noninvasive, thus eliminating any discomfort and pain associated with blood collection while also avoiding privacy issues associated with urine collection. Additionally, compared with blood, saliva contains a smaller quantity of proteins, therefore decreasing any potential risk of non-specic interference and hydrostatic interactions. Within blood, the protein concentration can vary over several orders of magnitude, with protein half-lives ranging from a few seconds to several months or longer. The composition of saliva, however, is not as complex or varying as serum, and should more accurately reect the current condition of the body at any given time. Ultimately, saliva may contain locally expressed proteins and other substances that can be used as indicators of diseases. These components, called biomarkers, can be closely related to an individuals health condition and can change greatly when diseases afict the body. Biomarker According to the National Institutes of Health, a biomarker is a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmaceutical responses to a therapeutic intervention.5 Generally speaking, a biomarker can be any biomolecule or specic characteristic, feature, or indicator of an alteration in any biological constitution and function that can objectively reect the state of a living organism.6 Criterion for biomarker  A major product of oxidative modication that may be implicated directly in the development of a disease;  A stable product, not susceptible to artefactual induction, not easy to lose, or not changeable during storage;  Representative of the balance between oxidative damage generation and clearance;  Determined by an analytical assay that is specic, sensitive, reproducible and robust;  Free of confounding and interference factors from dietary intake;

 Accessible in a target tissue or a valid surrogate tissue such as a leukocyte;  Detectable and measurable within the limits of detection of a reliable analytical procedure.7 The discoveryvalidationimplementation paradigm A biomarker must be veried and validated before it can have any impact or application on health risk assessment. The vericating process might be considered as a process that is conceptually similar to therapeutic drug evaluation. There are six prerequisites before a biomarker can be used in a clinical assay: (1) preclinical testing: developing in vitro or in animal models; (2) preliminary testing: developing preliminary assays on patient samples; (3) feasibility analysis: testing on a small group of patients to determine its ability to discriminate between healthy or diseased subjects; (4) validation of the accuracy of assays; (5) statistical analysis: determining in large patient populations; (6) post-approval reporting and testing. A general recommendation is that the validation effort should concentrate on those biomarkers directly involved in the causal pathway of disease, since the closer to the causal pathway the biomarker is, the more precisely it will predict disease.8 Saliva analysis In the last few years, remarkable efforts have been devoted to the identication of proteins in human and parotid saliva by using diverse proteomic approaches. High-resolution liquid separation is a critical component in both shotgun and random proteome analysis. Pre-fractionation of proteins using liquid-based separation techniques is often required for a comprehensive analysis. Separations can be performed based on the physiochemical properties of the interested protein using capillary isoelectric focusing (IEF),9 gel ltration liquid chromatography (LC), reversed-phase (RP) LC, strong cation exchange LC or ZOOM IEF. The fractions are collected and digested using proteolytic enzymes and the resulting peptides are analyzed with 1D-LC/MS/MS or 2D-LC/MS/MS, either online or ofine. The online 2D-LC separation uses a single capillary column packed with two types of LC separation media10 or an automatic column-switching technique. Free-ow electrophoresis can be coupled with RP-LC to greatly enhance the separation of peptides prior to MS/MS analysis.11 In other cases, investigators have used two-dimensional (2D) gel electrophoresis (GE) to separate protein components, followed by mass spectrometry (MS) to subsequently identify the peptides produced from in-gel digestion of the proteins of interest. This approach revealed that more than 300 proteins exist within saliva. When separations were performed using liquid chromatography

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(LC) instead of GE, the results from 2D-MS identied more than 1050 proteins in saliva.1214 Recently, surface-enhanced laser desorption/ionization time-of ight (SELDI-TOF) has also been utilized. This technique, which combines matrix-assisted laser desorption/ionization time-ofight mass spectrometry (MALDI-TOF-MS) with surface chromatography, enables rapid and high-throughput detection of critical proteins and peptides requiring only small amounts of non preprocessed sample.15 Finally, additional methodologies, such as high performance liquid chromatography/mass spectrometry (HPLC/MS), have proven to be useful in the evaluation of the smallest salivary proteins and peptides.16 Currently, the proteomic analysis of salivary biomarkers holds promise as a non-invasive method for identifying various diseases such as cancer, diabetes, and autoimmune diseases. These proling technologies may be integrated to achieve a more comprehensive analysis.

Table 2 Various salivary biomarkers signicantly altered in OSCC patients as compared with healthy controls.56 Biomarker IAP SCC CEA CA19-9 CA125 Cyfra 21-1 TPS RNS 8-OHdG IgG Sec IgA IGF MMP-2, MMP-11 LOH DNA hypermethylation IL8, IL 1B DUSP1 HA3 OAZ1 S100P SAT Biological function Apoptosis inhibitor Squamous cell carcinoma associated antigen Carcinogenic embryonic carcinogen Carcino-antigen Serum tumor marker Intermediate lament protein Tissue polypeptide specic antigen Reactive nitrogen species DNA damage marker Immunoglobulin Mucosal immunoglobin Growth factor Metalloproteinase Loss of heterozygosity-loss of specic chromosomal regions Gene inactivation Chemokine-mediator of inammatory response Cell proliferation regulator Oncogene Polyamine synthesis regulator Calcium binding protein, cell cycle and differentiation regulator Polyamine metabolism B2M,FTH1,G0S2,GADD45B,H3F3A,HSPC016, IER3,MAP2K3,PRG1,RGS257 Change Increased

Salivary biomarkers as a diagnostic tool for different disease Oral squamous cell carcinoma (OSCC) Oral squamous cell carcinoma (OSCC) is a common malignant tumor occurring with increasing frequency among individuals. The prevalence of OCSS has had a 5.3-fold increase for men and a 2-fold increase for women within the past two decades. The survival rate of oral cancer is 6080% when detected during its early stages; however, this number drops to 3040% when the cancer is diagnosed during the advanced stages.17 One pressing issue is the lack of a reliable early stage diagnostic marker for OSCC, meaning almost all OSCC cases are diagnosed when the cancer has developed well into the advanced stages. In addition, because OSCC has a very high recurrence rate, early identication and detection become essential for patient survival. Detection of OSCC is currently based on expert clinical examination and histological analysis of suspicious areas, but it may be undetectable in hidden sites. Therefore, sensitive and specic biomarkers for OSCC may be helpful for screening of high-risk patients.18 Several studies have developed methods for using salivary proteins as potential diagnostic markers for oral cancer. Increasing levels of saliva-soluble CD44 were shown in the majority of patients with OSCC and could be used to distinguish cancer from health with high specicity.19 Also, the concentration of three tumor markers: cytokeratin 19 fragment (Cyfra 21-1)20, tissue polypeptide antigen, and cancer antigen 125, were found signicantly elevated in the saliva of OSCC patients. Analysis of the concentrations of these three markers in both saliva and plasma yielded similar diagnostic results among OSCC patients.21 Also, the level of p53 autoantibodies measured in saliva was found to correlate with those levels in serum, potentially offering a specic method for detecting a subset of OSCC with p53 aberrations.22 However, these candidate biomarkers were discovered on an individual basis, limiting their potential for predicting OSCC. Table 2 present a selection of potential biomarkers found in OSCC patients. By using two-dimensional gel electrophoresis (2D-GE) and matrix-assisted laser desorption/ionization time-of-ight mass spectrometry (MALDI-TOF-MS), Cheng-Wen Lin analyzed the protein prole of pooled salivary samples from patients with oral squamous cell carcinoma (OSCC) and OSCC-free control subjects, nding elevated transferrin levels in the saliva of OSCC patients. Additionally, the magnitude of the salivary transferrin levels in OSCC patients strongly correlated with the size and stage of the tumor.23 Using laser-capture micro dissection, St John MAR4 have identied the expression of cellular gene that are uniquely associated with OSCC: interleukin (IL) 8. IL-8 has proven to be clinically signicant in

Others (salivary mRNA) Biomarker Carbonyls, lactate dehydrogenase, metalloproteinase-9 (MMP9) Ki67, Cyclin D1 (CycD1)58 8-Oxoguanine DNA glycosylase, Phosphorylated-Src, mammary serine protease inhibitor (Maspin)

Decreased

oral cancer diagnosis. Results showed higher concentrations of IL-8 in saliva among patients with OSCC. These cytokines may contribute to the pathogenesis of this disease, and have been linked with increased tumor growth and metastasis. As a salivary biomarker for early stage OSCC, IL-8 can be detected at 1.1 pM level using a surface immobilized sandwich assay technique.24 Therefore, the detection of IL-8 levels could prove to be a cost-effective tool in the diagnosis and monitoring of patients with OSCC. S. Shintani25 used surface-enhanced laser desorption/ionization time-of-ight mass spectrometry (SELDI-TOF) Protein Chip system to screen for differentially expressed proteins in the saliva samples. Shintani, suggested that Protein Chip analysis may provide a reliable screening test for early diagnosis of OSCC, with emphasis on the importance of truncated cystatin SA-I as an OSCC tumor biomarker. To conrm that truncated cystatin SA-I is an OSCC-specic protein, the expression levels in pre-and post-treatment saliva from OSCC patients were compared. Experiments performed on CM10 arrays showed an increased intensity of truncated cystatin SA-I in pre-treatment saliva samples compared to post-treatment samples. TNF-a has a salivary concentration approximately 30 pg/ml in oral cancer patients and 3 pg/ml in healthy individuals. Such a concentration discrepancy provides another potential biomarker for OSCC supervision. Most proteins found in saliva exist both in individuals with OSCC and in healthy individuals. However, 52 proteins were found to be present in OSCC patients only, and 29 proteins were found in healthy subjects only.26 The identity of each protein is listed in Table 3. Further validation on a larger patient cohort is required for these putative biomarkers.

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Table 3 Saliva proteins identied only from healthy control (compared with OSCC) subjects and only from OSCC (compared with healthy persons) by subtractive proteomics. Saliva proteins identied only from healthy control (compared with OSCC) subjects Clusterin Serine/arginine repetitive Matrix 1 Neurolament triplet H protein Uteroglobin Cask-interzcting protein 2 Actin-related protein 5 Utrophin Cornin A, B Sparc-like protein 1 Antileuko-proteinase 1 Airway trypsin-like protease Metalloproteinase inhibitor 1 9,42,43,86,172 kDa protein Hypothetical protein

Similar to heterogeneous nuclear ribonucleoprotein K Saliva proteins identied only from OSCC patients Catalase Azurocidin Beta-2-glycopr-otein 1 Enolase 1 Enolase 2 Enolase 3 Cathepsin G Peptidylprolyl isomerase A-like S-100P mprotein Splice isoform 2 of myeloperoxidase Epsilon globin Carbonic anhydrase 1 Calcium-bind-ing protein A12

Splice isoform 1 Of Desmoglein 3 ADAMTS-2 Similar to Ig Metaxin 1 isoform 1 gamma-3 C region Macrophage migration inhibitory factor Involucrin Vitamin D-binging protein Thioredoxin Squamous cell carcinoma antigen 2 Heat shock 70 KDa protein 1 Myeloblas-tin Similar to SEC14like protein 2 Hematopoi-etic lineage cell specic Transaldolase Brain acid Soluble Protein 1 Calgizzarin Haptoglobin-related protein Shroom-related protein Peroxisome biogenesis factor 1 Ras-related protein Rab-7 Putative S100 calcium-binding protein

Hemopexin Similar to Myomegalin Moesin Tumor-related protein

Calcyclin Phosphoglyc-erate kinase 1 Histone H1.2 CD59 glycoprotein

Peroxiredoxin 2 Triosephosphat-e isomerase Alpha-1-acid glycoprotein 1

Alpha enolase, lung specic Splice isoform 1 of myeloperoxidase Antibacterial protein FALL-39 11 kDa protein 16 kDa protein 57 kDa protein

Mac-2 binding protein Cytoplasmic antiproteinas-e 2 Muscarinic acetylcholine receptor M3

Splice isoform 1 of Transcription Intermediary factor 1-gamma

SH3 domain-binding glutamic acid-rich-like protein

Table 4 Possible salivary markers for periodontal diseases.59 Proteins He lactoferrin TIMP VEGF HGF Fibronectin Albumin Cystatins C, S, A, SN Neopterin a-2-Macroglobulin a-1-Antitrypsin Keratin C-reactive protein Complement C3 IL-6 EGF Defensin-1 Immunoglobulins IgA IgG IgM Enzymes Elastase Amylase Dipeptidylpeptidase Alanine aminopeptidase Arginase b-Glucuronidase Myeloperoxidase Lysozyme MMP-1 MMP-8(collagenase-2) MMP-960 Chitinase Cathepsin G Others PAF 8-OHdG Urate Ascorbate Cortisol Nitrite Glycosaminoglycans61 Cytokine TNF62 Hyaluronic acid Chondroitin sulphate Aspartate aminotransferase (AST) Alkaline phosphatase (ALP)63 Salivary sCD4464 MRP8 and MRP1465 8-Oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG)66 Cysteine67 3-Hydroxy fatty acids68 Protein carbonyl (PC)69

Because oral cancer cells are immersed in the salivary milieu, analysis of the salivary proteomes from OSCC patients is a promising approach to nding biomarkers for the disease. Saliva is an easily accessible uid compared with tissue obtained from biopsy. Therefore, a large number of saliva samples can be collected and analyzed, allowing for a robust study with sufcient statistical power to reveal true signatures for characteristics of the disease. Because OSCC is a complex disease resulting from an interdependent series of genetic alterations rather than a single decisive event, a combination of candidate protein markers can improve the sensitivity and specicity for OSCC detection. Periodontal disease Periodontitis is a group of inammatory diseases that is characterized by loss of connective tissue attachment and bone around the teeth in conjunction with the formation of periodontal pockets due

to the apical migration of the junctional epithelium.27 If left untreated, the disease continues with progressive bone destruction, leading to tooth mobility and subsequent tooth loss. Periodontal disease aficts over 50% of the adult population in the United States.28 The detection and utilization of molecular biomarkers correlating with periodontal disease would permit rapid and accurate diagnoses, dynamic monitoring of disease activity, and potentially more effective treatment. Some components of saliva proposed as disease markers include enzymes (alkaline phosphatase, esterase, glucuronidase, aminopeptidase), immunoglobulins (IgA, IgG), and steroid hormones. Many of these salivary components appeared to be useful biochemical markers. Saliva analysis, therefore, can be a cost-effective approach for monitoring the disease. Table 4 lists possible salivary biomarkers for periodontal diseases. Matrix metalloproteinase-8 (MMP-8) has been identied as a major tissue-destructive enzyme in periodontal disease. Consequently, MMP-8 is a promising candidate for diagnosing and

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assessing the progression of this episodic disease.29 Research from Herr et al. discusses the use of clinical point-of-care (POC) diagnostic that enables rapid quantitation of an oral disease biomarker in human saliva by using a monolithic disposable cartridge designed to operate in a compact analytical instrument. The microuidic method facilitates hands-free saliva analysis by integrating sample pretreatment (ltering, enrichment, mixing) with electrophoretic immunoassays to quickly measure analyte concentrations in minimally pretreated saliva samples. Using 20 ll of saliva, they could rapidly measure (<10 min) the MMP-8 collagen-cleaving enzyme concentration in saliva from healthy and periodontally diseased subjects.30 The microchip electrophoretic immunoassay (lCEI)

Figure 1A lCEI device layout. Fluid wells are labeled according to contents as follows: S: sample; B: buffer; SW: sample waste; BW: buffer waste; mAb: uorescently labeled monoclonal antibody to MMP-8. Inset shows a 40 brighteld image of the size-exclusion membrane.

diagnostic instrument used by Herr et al. relies on photolithographically fabricated molecular sieving gels to enrich samples and subsequently resolve uorescent antibodies from MMP-8 complex under native electrophoresis conditions. Additional schematics are provided in Fig. 1A and B. Recently, Lamster et al.31 found a signicant correlation between periodontal clinical parameters and salivary b-glucuronidase activity. In addition, the total number of white blood cells and neutrophils in blood was observed to be associated with the concentration of salivary b-glucuronidase. Conclusively, salivary b-glucuronidase activity was found to potentially reect the presence of periodontal disease. 8-Hydroxy-deoxyguanosine (8-OHdG) is a product of oxidative DNA damage following specic enzymatic cleavage after hydroxylation of the C8 atom in a guanine group. 8-OHdG is one of the most commonly used markers for evaluating the damage done by chronic inammatory diseases. Takane et al.32 collected saliva samples from patients with untreated periodontitis and healthy control subjects. Using ELISA, the mean value of 8-OHdG in the saliva samples of periodontally diseased subjects was determined to be higher than that of healthy subjects. Salivary 8-OHdG levels decreased in response to periodontal therapy and approached the mean control values. Fibronectin is a glycoprotein which mediates adhesion between cells. Consequently, bronectin is also involved in the processes of growth, migration and differentiation of the cells. A recent study claims that bronectin in saliva plays a regulatory role in porphyromonas gingivalis mbria-mediated pathogenesis in adult periodontal disease.33 ELISA tests further veried the claim, showing that the bronectin concentrations in saliva of adult periodontal patients was signicantly lower than that of healthy subjects. Immunoglobulin A (IgA) is the predominant immunoglobulin in saliva and is categorized into two subclasses: IgA1 and IgA2. IgA1 is predominantly in serum while IgA2 is found in higher concentrations in external secretions such as saliva. Hagewald et al.34 investigated the humoral IgA response in the saliva of aggressive periodontitis patients by measuring IgA subclasses and IgA antibodies reactive to microorganisms associated with periodontal disease. A signicantly lower concentration and secretion rate of total salivary IgA2 and IgA1 was found in the aggressive periodontitis group. Several studies have investigated the relationship between gingival crevicular uid (GCF) osteocalcin levels and periodontal disease. Kunimatsu et al.35 reported a positive correlation between GCF osteocalcin aminoterminal peptide levels and clinical parameters in a crosssectional study of periodontitis and gingivitis patients. Results from another study also revealed a signicantly higher level of total protein in the GCF of diseased teeth, suggesting the possibility of using total protein concentration as an indicator for periapical disease.36 All of the previously mentioned biomarkers are found in patients with periodontal disease. However, the validity of such species still needs to be examined with greater detail. Additionally, the discovery of more biomarkers of disease would always be welcome. Cancer Cancer is a major public health problem in many countries. Currently, one in four deaths in the United States is due to cancer.37 In addition, pancreatic cancer prognosis tends to be extremely poor, with one of the lowest survival rates among all cancers. Due to a lack of any visible symptoms during the early stages, detection capabilities are limited. New strategies and biomarkers for early detection are, therefore, desperately needed. Salivary biomarkers for a variety of cancers have been identied and may provide

Figure 1B On-chip sample enrichment. P1: the detection mixture is loaded against the size-exclusion membrane. P2: saliva sample is then loaded, resulting in coenrichment of saliva and aMMP-8 at the size-exclusion membrane. P3: an electric potential is applied across the membrane, causing the enriched species to elute into the separation channel, thus initiating the electrophoretic immunoassay. Subsequently, the electric potential is switched to omit the membrane from the current path. Current ow is indicated by i.

574 Table 5 Potential biomarkers for breast cancer detection.70 Blood biomarkers in saliva c-erbB-2 VEGF EGF CEA Biomarkers for breast cancer

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CSTA TPT1 IGF2BP1 GRM1 GRIK1 H6PD MDM4 S100A8 CA6(carbonic anhydrase VI) Psoriasin Cortisol and dehydro-epiandrosterone sulphate71 Cancer antigen 15-3 (CA15-3)

valuable diagnostic information. Zhang et al.38 have conducted a prospective sample collection and retrospective, double-blinded validation to evaluate the performance and translational utilities of salivary transcriptomic biomarkers for noninvasive detection of resectable pancreatic cancer. It was found that a combination of four messenger RNA biomarkers (KRAS, MBD3L2, ACRV1, and DPM1) in saliva supernant could differentiate pancreatic cancer patients from noncancer subjects. Recently, prostate cancer has become a major health issue in western countries. Excluding cutaneous malignancies, it stands as the most frequent malignant illness in men and the second leading cause of cancer-related mortality.39 Using microparticle enzyme immunoassay,40 free and total prostate-specic antigen (PSA) levels and the free/total (f/t) ratio in the saliva could be compared to those in the serum of normal individuals, patients with benign prostatic hyperplasia (BPH), and prostate cancer. While there was a signicant difference between mean serum and salivary levels of free and total PSA, the f/t ratio in both saliva and serum were very close among normal subjects. Additionally, glycoprotein biomarkers for human cancers, such as prostate-specic antigen, protein c-erbB-2, cancer antigen (CA) 125, 19-9 and 15-3, and carcinoembryonic antigen (CEA), have also been detected in human saliva. Saliva testing of glycoprotein biomarkers may be another promising approach to human cancer detection.41 Breast cancer is the most commonly diagnosed form of cancer and the leading cause of cancer death in women today. Clinically useful biomarkers for early detection of breast cancer could lead to a signicant reduction in mortality rates. The biomarkers for breast cancer are listed in Table 5. The protein c-erbB-2, also known as Her2/neu, is a prognostic breast cancer marker assayed in tissue biopsies from women diagnosed with malignant tumors. Present studies suggest that soluble fragments of the c-erbB-2 oncogene may be released from the cell surface and become detectable in patients with carcinoma of the breast. To determine the diagnostic utility of this oncogene, the soluble form of the c-erbB-2 protein was assayed in the saliva and serum using ELISA in three different groups of women. Findings showed the presence of the c-erbB-2 protein in both the saliva and serum of all three groups of women. Moreover, salivary and serum levels of c-erbB-2 in the cancer patients were signicantly higher than the salivary and serum levels of healthy control subjects and benign tumor patients. These results suggest that the cerbB-2 protein may have potential use in the initial detection and/or follow-up screening of breast cancer in women.42 Total protein concentration, lipid peroxidation (LPO) levels and pH values in the saliva of breast cancer patients were also found lower than those in the saliva of healthy individuals. Tissue factor (TF), known as thromboplastin or Factor III, is considered to be a major regulator of normal hemostasis and thrombosis. Studies have shown that TF activity is higher in breast cancer patients than

that in the control group, but the difference may not be statistically signicant.43 Another study also showed that the levels of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and carcinoembryonic antigen (CEA) in the saliva were signicantly elevated in cancer patients. Conclusively, saliva is believed to be a novel avenue for tumor marker research; and with additional efforts and developments, saliva analysis may be a useful supplement to current methods of breast cancer detection.44 Tongue cancer is amongst the most common and fatal types of cancers in the world. Despite advances in cancer detection and treatment, the prognosis of squamous cell carcinoma of the tongue (TSCC) has not greatly improved within the last few decades and remains one of the most common and fatal head and neck cancers worldwide. Studies from Masood et al.45 examined the levels of IL1a, IL-6, IL-8, VEGF-a and TNF-a in saliva using quantitative ELISA in three different groups of individuals (endophytic TSCC patients, exophytic TSCC patients and healthy subjects). Research shows that all ve cytokines were elevated in the endophytic TSCC group compared to the other groups. IL-1a, IL-6, TNF-a and VEGF were also elevated in the exophytic TSCC group compared to the control group. Salivary levels of IL-1a, IL-6, IL-8, VEGF-a and TNF-a, could serve as potential biomarkers for cancer screening and early detection and can also be used to identify the progression of TSCC. Another relevant work has demonstrated that salivary adenosine deaminase (ADA) might be used as a diagnostic tool for early detection of squamous cell carcinoma of the tongue.46 Sjgrens syndrome Sjgrens syndrome (SS) is a chronic autoimmune disease, being characterized by epithelial cell destruction due to peri-epithelial B and T lymphocytes inltrating and targeting multiple organs, particularly the moisture producing exocrine glands. Because salivary and lachrymal glands are involved, dry mouth (xerostomia) and dry eyes (xerophtalmia) represent the typical clinical symptoms of the disease. Sjgrens syndrome is one of the three most common autoimmune disorders. Many changes in SS salivary constituents have been described previously (see Table 6), suggesting that saliva could be used to diagnose the syndrome. Saliva samples gathered from individuals with SS show increased concentrations of Na+, Cl, IgG, lysozyme, matrix metalloproteinase (MMP)-2 and MMP-9 in parotid saliva, as well as increased concentrations of lactoferrin, IgA, b2-microglobulin, albumin in both parotid and whole saliva, and increased concentrations of kallikrein and cystatins C and S in whole saliva. It has also been indicated that the SS salivary protein prole, contains an increased number of inammatory proteins and decreased number of acinar proteins. MMP-2, MMP-9, TIMP-1, and TIMP-2 levels were measured using enzyme-linked immunosorbent assay (ELISA) and sandwich enzyme immunoassay (sandwich EIA). The study found that the ratio of MMP-9/TIMP-1 and MMP-9 levels in the saliva were signicantly higher in primary SS (pSS) patients than those in healthy subjects. The results suggest that an increase in the overall MMP9/TIMP-1 ratio, as opposed to simply an increase in MMP-9, in pSS patients saliva strongly correlates with the destruction of glandular and salivary duct tissues.47 Analyses of parotid and whole saliva using ELISA show the signicant increase in lactoferrin and b2-microglobulin levels among SS patients. b2-Microglobulin, a light-chain molecule categorized as a major histocompatibility complex class I antigen, is present on the membrane surface of many nucleated cells, including inltrating lymphocytes and salivary gland epithelium. Increased levels of this protein in SS saliva may, therefore, relate to salivary gland inammatory activity rather than lymphocyte numbers.48 Lactoferrin is a product of intercalated ductal cells and scattered

J. Liu, Y. Duan / Oral Oncology 48 (2012) 569577 Table 6 Alterations of salivary proteins in primary SS.72 Salivary component Change Salivary component a-Amylase Carbonic anhydrase VI Decreased (1) Lipocalin 1precursor (2) Calgranulin B (3) Phosphatidyl ethanolamine binding protein Increased Prostaglandin E2 ThromboxaneB2 [TxB2] Increased Neopterin75 IFN-a76 Increased Increased Proline-rich Proteins (PRPs) Prolactin-Inducible Protein Precursor (PIP) Decreased Cystatins (S,SN) Lactoferrin b-2microglobulin Increased Lysozyme C Cystatin C

575

lg k light chain Polymeric Ig receptor Increased MMP-9/TIMP-1 Immunoglobulin A,G

Change Salivary marker Change Salivarymarker Change Change

Decreased Interleukin-6 (IL-6) Hyaluronic acid (HA) Increased Gamma-glutamyl-tra-nsferase (GGT)77 Increased Increased

Increased Increased Soluble interleukin-2 receptor (sIL-2R)73 Protein-conjugated acrolein74 Increased

acinar cells in the parotid gland. Previous studies of SS saliva have reported increases in lactoferrin levels without a clear association to the amount of lymphocytic inltration.49 Since lactoferrin levels increase in other diseases pertaining to the salivary glands, such as parotitis50 and diabetes,51 it cannot be used independently to diagnose SS. T.J. Kramer52 quantied C-X-C motif chemokine 13 (CXCL13) with real-time polymerase chain reaction and enzyme-linked immunosorbent assay at various stages of SS disease using primary SS (pSS) and secondary SS (sSS) models. The results show that CXCL13 transcription and protein levels increase with disease severity in salivary tissue and serum, respectively. Moreover, CXCL13 colocalizes with lymphocytes in salivary tissue. At the late stages of SS, increasing levels of CXCL13 in saliva correlate with that of blood. Therefore, the therapeutic targeting of CXCL13 may provide an innovative approach for managing SS disease. In SS patients, the salivary concentrations of immunoglobulin IgA and IgG, lactoferrin, b2-microglobulin, eicosanoids (prostaglandin E2 and Thromboxane B2 [TxB2]), interleukin-6 (IL-6), and hyaluronic acid (HA) were all elevated compared with control groups. Other studies have even suggested using an increase in salivary IgA as a criterion for the diagnosis of SS. Furthermore, saliva proteome analysis of pSS patients broadly links pSS disease to an increase in inammatory proteins and a decrease in acinar protein compared to non-SS subjects.53,54 Current diagnosis of pSS requires a salivary gland biopsy. However, validation of newly discovered biomarkers could result in a noninvasive method of pSS diagnosis in the near future. Although a wide range of potential biomarkers for SS detection exists, there are still many obstacles to overcome. Several constituents of saliva have been evaluated within the last 20 years, but none have been specic or sensitive enough for the diagnosis of SS. Saliva, which is produced by three major and numerous minor glands, has great variations in both ow and control among individuals, and, at times, even in the same individual under diverse conditions. Another problem that arises is the use of different types of saliva in various studies. The way in which saliva is collected, either stimulated or unstimulated, can signicantly affect saliva composition. Furthermore, because the rate of salivary ow from the submandibular/sublingual glands in SS patients is slower than that of healthy individuals, collecting whole saliva versus saliva from the parotid/submandibular glands can affect the outcome of the study. Lastly, the criteria for selecting SS patients have changed within the past few decades. While earlier works have used various criteria for enrolling SS patients, a publication from the European Community in 1993 has tightened the standards for SS diagnosis. These guidelines, which have been accepted by most rheumatologists, should be used systematically for future studies.

Prospect Saliva has great potential for the surveillance of general body health and disease. To reach the above goal through saliva-based diagnostics, a new form of miniaturization technology known as lab-on-a-chip may help through detecting multiple compounds in parallel and allows for simultaneous assessment of multiple disease conditions. This technology also provides possibility for pointof-care diagnostics. Moreover, because lab-on-a-chip technology allows for a personal and private diagnosis outside of the laboratory, such as at home, it may further enhance healthcare delivery, reduce health disparities, and improve access to care. This new technology in association with the saliva-based approach, which is non-invasive, inexpensive, easier, and safer than approaches based on serum or urine, can signicantly impact disease diagnostics.2 Identifying disease diagnostic markers and successfully translating research efforts from the laboratory into clinic is the greatest challenge for salivary diagnostics. Candidate biomarkers need to be extensively tested and studied, as much more validation is required. Proteome analysis has provided signicant insight, but faces many obstacles as well. For instance, the sampling efciency of LC-MS/MS varies from one experiment to another, with some of the target biomarkers identied based on single-peptide assignment. Furthermore, due to a dramatic abundance of amylase in human saliva, effective removal of salivary amylases prior to proteome analysis is both necessary and challenging. Clearly, it is challenging to translate candidate biomarkers from proteomic investigations into real-world diagnostic or prognostic applications. However, new diagnostic tools such as nucleic acid and protein microarrays and microuidics are under development for assessment and comprehensive screening of biomarkers. If appropriately validated on larger patient cohorts, testing of candidate biomarkers coupled with microuidic devices may become a powerful tool for oral cancer diagnosis in the future. Device cartridges, which can simplify assay operation and improve assay sensitivity by integrating saliva pretreatment (mixing, incubation, and enrichment) with subsequent quantitative analysis, may also be an attractive option. Solid-phase microextraction (SPME) has gained widespread acceptance as a means of analyte-matrix separation and sample preconcentration. Furthermore, it is compatible with other separation/detection techniques such as gas chromatography and/or high performance liquid chromatography, while providing linear results for a wide range of concentration.55 By taking into consideration the proper stationary-phase coating layer, it is possible to reach a low detection limit with minimized matrix interference.

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J. Liu, Y. Duan / Oral Oncology 48 (2012) 569577 15. Schipper R, Loof A, de Groot J, Harthoorn L, Dranseld E, van Heerde W. SELDITOF-MS of saliva: methodology and pre-treatment effects. J Chromatogr B Analyt Technol Biomed Life Sci 2007;847(1):4553. 16. Hardt M, Thomas LR, Dixon SE, Newport G, Agabian N, Prakobphol A, et al. Toward dening the human parotid gland salivary proteome and peptidome: identication and characterization using 2D SDS-PAGE, ultraltration, HPLC, and mass spectrometry. Biochemistry 2005;44(8):288599. 17. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA-Cancer J Clin 2005;55(2):74108. 18. Hou XL, Deng DL, Wu X, Lv Y, Zhang JY. Simultaneous stacking of cationic and anionic compounds in single run capillary zone electrophoresis by two-end eld amplied sample injection. J Chromatogr A 2010;1217(35):56227. 19. Franzmann EJ, Reategui EP, Pedroso F, Pernas FG, Karakullukcu BM, Carraway KL, et al. Soluble CD44 is a potential marker for the early detection of head and neck cancer. Cancer Epidemiol Biomarkers Prev 2007;16(7):134855. 20. Zhong LP, Zhang CP, Zheng JW, Li J, Chen WT, Zhang ZY. Increased Cyfra 211 concentration in saliva from primary oral squamous cell carcinoma patients. Arch Oral Biol 2007;52(11):107987. 21. Nagler R, Bahar G, Shpitzer T, Feinmesser R. Concomitant analysis of salivary tumor markers A new diagnostic tool for oral cancer. Clin Cancer Res 2006;12(13):397984. 22. Tavassoli M, Brunel N, Maher R, Johnson NW, Soussi T. p53 antibodies in the saliva of patients with squamous cell carcinoma of the oral cavity. Int J Cancer 1998;78(3):3901. 23. Jou Y-J, Lin C-D, Lai C-H, Chen C-H, Kao J-Y, Chen S-Y, et al. Proteomic identication of salivary transferrin as a biomarker for early detection of oral cancer. Anal Chim Acta 2010;681(12):418. 24. Tan W, Sabet L, Li Y, Yu T, Klokkevold PR, Wong DT, et al. Optical protein sensor for detecting cancer markers in saliva. Biosens Bioelectron 2008;24(2):26671. 25. Shintani S, Hamakawa H, Ueyama Y, Hatori M, Toyoshima T. Identication of a truncated cystatin SA-I as a saliva biomarker for oral squamous cell carcinoma using the SELDI ProteinChip platform. Int J Oral Maxillofac Surg 2010;39(1):6874. 26. Shen Hu, Martha Arellano, Boontheung Pinmanee. Salivary proteomics for oral cancer biomarker discovery. Clin Cancer Res 2008;14:624652. 27. Ozmeric N. Advances in periodontal disease markers. Clin Chim Acta 2004;343(12):116. 28. Albandar JM. Periodontal diseases in North America. Periodontol 2000 2002;29(1):3169. 29. Kiili M, Cox SW, Chen HW, Wahlgren J, Maisi P, Eley BM, et al. Collagenase-2 (MMP-8) and collagenase-3 (MMP-13) in adult periodontitis: molecular forms and levels in gingival crevicular uid and immunolocalisation in gingival tissue. J Clin Periodontol 2002;29(3):22432. 30. Herr AE, Hatch AV, Throckmorton DJ, Tran HM, Brennan JS, Giannobile WV, et al. Microuidic immunoassays as rapid saliva-based clinical diagnostics. Proc Natl Acad Sci U S A 2007;104(13):526873. 31. Lamster IB, Kaufman E, Grbic JT, Winston LJ, Singer RE. Beta-glucuronidase activity in saliva: relationship to clinical periodontal parameters. J Periodont 2003;74(3):3539. 32. Takane M, Sugano N, Iwasaki H, Iwano Y, Shimizu N, Ito K. New biomarker evidence of oxidative DNA damage in whole saliva from clinically healthy and periodontally diseased individuals. J Periodont 2002;73(5):5514. 33. Murakami Y, Hanazawa S, Tanaka S, Iwahashi H, Kitano S, Fujisawa S. Fibronectin in saliva inhibits Porphyromonas gingivalis mbria-induced expression of inammatory cytokine gene in mouse macrophages. FEMS Immunol Med Microbiol 1998;22(3):25762. 34. Hagewald S, Bernimoulin JP, Kottgen E, Kage A. Salivary IgA subclasses and bacteria-reactive IgA in patients with aggressive periodontitis. J Periodontal Res 2002;37(5):3339. 35. Kunimatsu K, Mataki S, Tanaka H, et al. A cross-sectional study on osteocalcin levels in gingival crevicular uid from periodontal patients. J Periodont 1993;64(9):8659. 36. Burgener B, Ford AR, Situ H, Fayad MI, Hao JJ, Wenckus CS, et al. Biologic markers for odontogenic periradicular periodontitis. J Endodont 2010;36(8):130710. 37. Jemal A, Siegel R, Ward E, Murray T, Xu JQ, Thun MJ. Cancer statistics, 2007. CACancer J Clin 2007;57(1):4366. 38. Zhang L, Farrell JJ, Zhou H, Elashoff D, Akin D, Park NH, et al. Salivary transcriptomic biomarkers for detection of resectable pancreatic cancer. Gastroenterology 2010;138(3):U194949. 39. Jemal A, Tiwari RC, Murray T, Ghafoor A, Samuels A, Ward E. Cancer statistics. CA-Cancer J Clin 2004;54(1):829. 40. Turan T, Demir S, Aybek H, Atahan O, Tuncay OL, Aybek Z. Free and total prostate-specic antigen levels in saliva and the comparison with serum levels in men. Eur Urol 2000;38(5):5504. 41. Hu S, Loo JA, Wong DT. Human saliva proteome analysis and disease biomarker discovery. 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Conclusion Since the collection of saliva is less invasive than that of blood for clinical analysis, it has become an attractive diagnostic uid for disease. A noninvasive collection method not only simplies a patients ability to take repeated samples for long-term disease monitoring, but signicantly reduces the pain and anxiety that is typically associated with blood tests. Unlike blood sample, which is prone to clotting, saliva is much easier to handle and requires less pre-analysis manipulation. Moreover, secretions from glands within the oral cavity contain proteins are uniquely associated with saliva. Therefore, compared with serum based biomarkers, salivary proteins may be a more sensitive and specic indicator for certain oral diseases. However, some relevant problems can not be ignored. Many putative biomarkers in saliva were independently discovered and must be further validated before clinical availability for because all the current individual markers are not sensitive and specic enough to meet strict diagnostic criteria. Also, most current published results are still preliminary and most of these studies were conducted in a very small number of samples with no specic marker being carefully validated. One possible way to overcome the limitations of single disease biomarkers is to set up a biomarker array to enhance the reproducibility and specicity for disease monitoring through simultaneous measurements of multiple biomarkers. Although large-scale, quantitative, high-throughput proteomics technologies are still in its very early stages, there is a likelihood that new breakthroughs will be made in the future. We highly expect that salivary diagnostics will become a complementary tool in routine health monitoring and early detection of diseases in the near future. Conict of interest statement None declared. References
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