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Oral Oncology 48 (2012) 569577

Contents lists available at SciVerse ScienceDirect

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

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

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 fluid, 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 define 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.

Introduction
Early detection of disease plays a significant 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 field of healthcare research. Saliva, a multi-constituent oral fluid 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 significant 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 fulfilled: (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 specificity of biomarkers
through a series of continuous developments.2
Saliva profile
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

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J. Liu, Y. Duan / Oral Oncology 48 (2012) 569577

Table 1
Salivary proteins.3
Salivary protein

Origin

Functions

Total proteins
a-Amylase
Albumin
Cystatins group
Hystatin
Secretory IgA
Lactoferrin
Lysozyme
Mucins group
PRPs

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

Statherin
Transferrin

Plasma

Ca++ binding

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

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-specific
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 reflect 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 afflict 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 specific characteristic, feature, or indicator of an alteration in any biological constitution and function that can objectively reflect the state of a
living organism.6
Criterion for biomarker
" A major product of oxidative modification 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 specific, 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 verified and validated before it can have
any impact or application on health risk assessment. The verificating 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 identification 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
filtration 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
offline. 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-flow 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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J. Liu, Y. Duan / Oral Oncology 48 (2012) 569577

(LC) instead of GE, the results from 2D-MS identified more than
1050 proteins in saliva.1214
Recently, surface-enhanced laser desorption/ionization time-of
flight (SELDI-TOF) has also been utilized. This technique, which
combines matrix-assisted laser desorption/ionization time-offlight 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 profiling technologies may be
integrated to achieve a more comprehensive analysis.

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 identification 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 specific 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 specificity.19 Also, the concentration of three tumor markers: cytokeratin 19 fragment (Cyfra 21-1)20, tissue polypeptide antigen, and cancer antigen 125, were found significantly
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 specific 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-flight mass spectrometry (MALDI-TOF-MS), Cheng-Wen Lin analyzed the protein
profile of pooled salivary samples from patients with oral squamous cell carcinoma (OSCC) and OSCC-free control subjects, finding 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 identified the expression of cellular gene that are uniquely associated with
OSCC: interleukin (IL) 8. IL-8 has proven to be clinically significant in

Table 2
Various salivary biomarkers significantly altered in OSCC patients as compared with
healthy controls.56
Biomarker

Biological function

IAP
SCC

Apoptosis inhibitor
Squamous cell carcinoma associated
antigen
Carcinogenic embryonic carcinogen
Carcino-antigen
Serum tumor marker
Intermediate filament protein
Tissue polypeptide specific antigen
Reactive nitrogen species
DNA damage marker
Immunoglobulin
Mucosal immunoglobin
Growth factor
Metalloproteinase
Loss of heterozygosity-loss of specific
chromosomal regions
Gene inactivation

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

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)

Chemokine-mediator of inflammatory
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

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-flight 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 confirm that truncated cystatin SA-I is an OSCC-specific
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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J. Liu, Y. Duan / Oral Oncology 48 (2012) 569577

Table 3
Saliva proteins identified only from healthy control (compared with OSCC) subjects and only from OSCC (compared with healthy persons) by subtractive proteomics.
Saliva proteins identified only from healthy control (compared with OSCC) subjects
Clusterin

Uteroglobin

Utrophin

Serine/arginine repetitive Matrix 1

Cask-interzcting protein 2

Neurofilament triplet H protein

Actin-related protein 5

Splice isoform 1 Of Desmoglein 3


ADAMTS-2
Similar to Ig
Metaxin 1 isoform 1
gamma-3 C region
Macrophage migration inhibitory factor

Similar to heterogeneous nuclear ribonucleoprotein K


Saliva proteins identified only from OSCC patients
Catalase
Azurocidin
Beta-2-glycopr-otein 1
Enolase 1 Enolase 2 Enolase 3

Cornifin A, B

Involucrin
Vitamin D-binging
protein
Thioredoxin
Squamous cell
carcinoma antigen
2
Heat shock 70 KDa
protein 1
Myeloblas-tin

Cathepsin G
Peptidylprolyl isomerase A-like

S-100P mprotein
Splice isoform 2 of
myeloperoxidase

Peroxiredoxin 2

Epsilon globin

Triosephosphat-e isomerase

Carbonic anhydrase 1

Alpha-1-acid glycoprotein 1

Calcium-bind-ing protein A12

Similar to SEC14like protein 2

Splice isoform 1 of Transcription


Intermediary factor 1-gamma

SH3 domain-binding glutamic


acid-rich-like protein

Hematopoi-etic
lineage cell specific

Transaldolase
Brain acid Soluble
Protein 1
Calgizzarin
Haptoglobin-related
protein

Sparc-like protein 1
Antileuko-proteinase 1
Airway trypsin-like
protease

Hemopexin
Similar to Myomegalin
Moesin
Tumor-related protein

Shroom-related
protein
Peroxisome
biogenesis factor 1
Ras-related protein
Rab-7

Alpha enolase, lung


specific
Splice isoform 1 of
myeloperoxidase
Antibacterial protein
FALL-39

Putative S100
calcium-binding
protein

11 kDa protein 16 kDa


protein 57 kDa protein

Metalloproteinase
inhibitor 1
9,42,43,86,172 kDa
protein
Hypothetical protein

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

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

Table 4
Possible salivary markers for periodontal diseases.59
Proteins

Immunoglobulins

Enzymes

Others

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

IgA
IgG
IgM

Elastase
Amylase
Dipeptidylpeptidase
Alanine aminopeptidase
Arginase
b-Glucuronidase
Myeloperoxidase
Lysozyme
MMP-1
MMP-8(collagenase-2)
MMP-960
Chitinase
Cathepsin G

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 finding biomarkers for the disease. Saliva is an easily accessible fluid compared with tissue obtained from biopsy.
Therefore, a large number of saliva samples can be collected and
analyzed, allowing for a robust study with sufficient 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 specificity for OSCC detection.
Periodontal disease
Periodontitis is a group of inflammatory 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 afflicts 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 identified as a
major tissue-destructive enzyme in periodontal disease. Consequently, MMP-8 is a promising candidate for diagnosing and

J. Liu, Y. Duan / Oral Oncology 48 (2012) 569577

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 microfluidic
method facilitates hands-free saliva analysis by integrating sample
pretreatment (filtering, 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#:
fluorescently labeled monoclonal antibody to MMP-8. Inset shows a 40$ brightfield image of the size-exclusion membrane.

573

diagnostic instrument used by Herr et al. relies on photolithographically fabricated molecular sieving gels to enrich samples
and subsequently resolve fluorescent 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 significant 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 reflect the presence of periodontal disease.
8-Hydroxy-deoxyguanosine (8-OHdG) is a product of oxidative
DNA damage following specific 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 inflammatory 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, fibronectin is also involved in the processes of
growth, migration and differentiation of the cells. A recent study
claims that fibronectin in saliva plays a regulatory role in porphyromonas gingivalis fimbria-mediated pathogenesis in adult
periodontal disease.33 ELISA tests further verified the claim, showing that the fibronectin concentrations in saliva of adult periodontal patients was significantly 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 significantly 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 fluid (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 significantly
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

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 flow is indicated by i.

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 identified and may provide

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J. Liu, Y. Duan / Oral Oncology 48 (2012) 569577

Table 5
Potential biomarkers for breast cancer detection.70
Blood biomarkers in saliva

Biomarkers for breast cancer

c-erbB-2
VEGF
EGF
CEA

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-specific 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 significant 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-specific 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 significant 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 significantly
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
significant.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 significantly 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 five 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 infiltrating 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 profile, contains an increased number of inflammatory 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 significantly 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 significant 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 infiltrating lymphocytes and salivary gland epithelium. Increased levels of this protein in SS saliva may, therefore, relate to salivary
gland inflammatory activity rather than lymphocyte numbers.48
Lactoferrin is a product of intercalated ductal cells and scattered

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J. Liu, Y. Duan / Oral Oncology 48 (2012) 569577


Table 6
Alterations of salivary proteins in primary SS.72
Salivary
component

a-Amylase Carbonic anhydrase VI

Proline-rich Proteins (PRPs) Prolactin-Inducible


Protein Precursor (PIP)

Lactoferrin b-2microglobulin

lg k light chain Polymeric Ig


receptor

Change
Salivary
component

Decreased
(1) Lipocalin 1precursor
(2) Calgranulin B
(3) Phosphatidyl
ethanolamine
binding protein
Increased
Prostaglandin E2
ThromboxaneB2 [TxB2]
Increased
Neopterin75
IFN-a76
Increased
Increased

Decreased
Cystatins (S,SN)

Increased
Lysozyme C
Cystatin C

Increased
MMP-9/TIMP-1
Immunoglobulin A,G

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

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

Change
Salivary
marker
Change
Salivarymarker
Change
Change

Increased
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 infiltration.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 quantified 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 inflammatory 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 specific or sensitive enough for the diagnosis of
SS. Saliva, which is produced by three major and numerous minor
glands, has great variations in both flow 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 significantly affect saliva
composition. Furthermore, because the rate of salivary flow 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 significantly 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 significant insight, but
faces many obstacles as well. For instance, the sampling efficiency
of LC-MS/MS varies from one experiment to another, with some of
the target biomarkers identified 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 microfluidics are under development for
assessment and comprehensive screening of biomarkers. If appropriately validated on larger patient cohorts, testing of candidate
biomarkers coupled with microfluidic 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.

576

J. Liu, Y. Duan / Oral Oncology 48 (2012) 569577

Conclusion
Since the collection of saliva is less invasive than that of blood
for clinical analysis, it has become an attractive diagnostic fluid
for disease. A noninvasive collection method not only simplifies a
patients ability to take repeated samples for long-term disease
monitoring, but significantly 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 specific 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 specific 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 specific 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 specificity 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.
Conflict of interest statement
None declared.
References
1. Streckfus CF, Dubinsky WP. Proteomic analysis of saliva for cancer diagnosis.
Expert Rev Proteomics 2007;4(3):32932.
2. Lee YH, Wong DT. Saliva: an emerging biofluid for early detection of diseases.
Am J Dent 2009;22(4):2418.
3. Chiappin S, Antonelli G, Gatti R, De Palo EF. Saliva specimen: a new laboratory
tool for diagnostic and basic investigation. Clin Chim Acta 2007;383(1
):3040.
4. St John MAR, Li Y, Zhou XF, Denny P, Ho CM, Montemagno C, et al. Interleukin 6
and interleukin 8 as potential biomarkers for oral cavity and oropharyngeal
squamous
cell
carcinoma.
Arch
Otolaryngol
Head
Neck
Surg
2004;130(8):92935.
5. Ilyin SE, Belkowski SM, Plata-Salamn CR. Biomarker discovery and validation:
technologies and integrative approaches. Trends Biotechnol 2004;22(8):4116.
6. Silberring J, Ciborowski P. Biomarker discovery and clinical proteomics. TracTrend Anal Chem 2010;29(2):12840.
7. Griffiths HR, Moller L, Bartosz G, Bast A, Bertoni-Freddari C, Collins A, et al.
Biomarkers. Mol Aspects Med 2002;23(13):101208.
8. Bonassi S, Neri M, Puntoni R. Validation of biomarkers as early predictors of
disease. Mutat Res-Fund Mol M 2001;480481:34958.
9. Guo T, Rudnick PA, Wang WJ, Lee CS, Devoe DL, Balgley BM. Characterization of
the human salivary proteome by capillary isoelectric focusing/nanoreversedphase liquid chromatography coupled with ESI-tandem MS. J Proteome Res
2006;5(6):146978.
10. Washburn MP, Wolters D, Yates JR. Large-scale analysis of the yeast proteome
by multidimensional protein identification technology. Nat Biotechnol
2001;19(3):2427.
11. Xie HW, Rhodus NL, Griffin RJ, Carlis JV, Griffin TJ. A catalogue of human saliva
proteins identified by free flow electrophoresis-based peptide separation and
tandem mass spectrometry. Mol Cell Proteomics 2005;4(11):182630.
12. Huang CM. Comparative proteomic analysis of human whole saliva. Arch Oral
Biol 2004;49(12):95162.
13. Amado FML, Vitorino RMP, Domingues P, Lobo MJC, Duarte JAR. Analysis of the
human saliva proteome. Expert Rev Proteomics 2005;2(4):52139.
14. Ghafouri B, Tagesson C, Lindahl M. Mapping of proteins in human saliva using
two-dimensional gel electrophoresis and peptide mass fingerprinting.
Proteomics 2003;3(6):100315.

15. Schipper R, Loof A, de Groot J, Harthoorn L, Dransfield 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 defining the human parotid gland salivary proteome and peptidome:
identification and characterization using 2D SDS-PAGE, ultrafiltration, 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
field amplified 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
identification 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. Identification 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 fluid 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. Microfluidic 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 fimbria-induced
expression of inflammatory 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 fluid 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-specific 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. Expert Rev Proteomics 2007;4(4):5318.
42. Streckfus C, Bigler L, Dellinger T, Dai XL, Kingman A, Thigpen JT. The presence of
soluble c-erbB-2 in saliva and serum among women with breast carcinoma: a
preliminary study. Clin Cancer Res 2000;6(6):236370.
43. Emekli-Alturfan E, Demir G, Kasikci E, Tunali-Akbay T, Pisiriciler R, Caliskan E,
et al. Altered biochemical parameters in the saliva of patients with breast
cancer. Tohoku J Exp Med 2008;214(2):8996.
44. Brooks MN, Wang JG, Yang L, Zhang R, Elashoff D, Wong DT. Salivary
protein factors are elevated in breast cancer patients. Mol Med Rep
2008;1(3):3758.

J. Liu, Y. Duan / Oral Oncology 48 (2012) 569577


45. Alexis Korostoff Lindsay Reder, Masood Rizwan, Sinha Uttam K. The role of
salivary cytokine biomarkers in tongue cancer invasion and mortality. Oral
Oncol 2011;47(4):2827.
46. Rai B, Kaur J, Jacobs R, Anand SC. Adenosine deaminase in saliva as a diagnostic
marker of squamous cell carcinoma of tongue. Clin Oral Investig
2011;15(3):3479.
47. Asatsuma M, Ito S, Watanabe M, Takeishi H, Nomura S, Wada Y, et al. Increase
in the ratio of matrix metalloproteinase-9 to tissue inhibitor of
metalloproteinase-1 in saliva from patients with primary Sjgrens syndrome.
Clin Chim Acta 2004;345(12):99104.
48. Tishler M, Yaron I, Shirazi I, Yaron M. Saliva: an additional diagnostic tool in
Sjogrens syndrome. Semin Arthritis Rheum 1997;27(3):1739.
49. Konttinen YT, Kulomaa M, Malmstrm M, Kilpi A, Reitamo S. Lactoferrin in
Sjgrens syndrome. Arthritis Rheum 1984;27(4):4627.
50. Tabak L, Mandel ID, Karlan D, Baurmash H. Alterations in lactoferrin in salivary
gland disease. J Dent Res 1978;57(1):437.
51. Dodds MWJ, Yeh CK, Johnson DA. Salivary alterations in type 2 (non-insulindependent) diabetes mellitus and hypertension. Community Dentist Oral
Epidemiol 2000;28(5):37381.
52. Jill M, Kramer DDS, Thomas L. CXCL13 in SJGren syndrome: a novel biomarker
of disease. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;110(6):e15.
53. Ryu OH, Atkinson JC, Hoehn GT, Illei GG, Hart TC. Identification of parotid
salivary biomarkers in Sjogrens syndrome by surface-enhanced laser
desorption/ionization time-of-flight mass spectrometry and two-dimensional
difference gel electrophoresis. Rheumatology 2006;45(9):107786.
54. Hu S, Wang JH, Meijer J, Leong S, Xie YM, Yu TW, et al. Salivary proteomic and
genomic biomarkers for primary Sjogrens syndrome. Arthritis Rheum
2007;56(11):3588600.
55. Ashwini Kumar, Ashok Kumar Malik, Matysik FM. Analysis of biological
samples using solid-phase microextraction. Bioanal Rev 2009;1:3555.
56. Nagler RM. Saliva as a tool for oral cancer diagnosis and prognosis. Oral Oncol
2009;45(12):100610.
57. Li Y, St John MAR, Zhou X, Kim Y, Sinha U, Jordan RCK, et al. Salivary
transcriptome diagnostics for oral cancer detection. Clin Cancer Res
2004;10(24):844250.
58. Shpitzer T, Hamzany Y, Bahar G, Feinmesser R, Savulescu D, Borovoi I, et al.
Salivary analysis of oral cancer biomarkers. Br J Cancer 2009;101(7):11948.
59. Ozmeric N. Advances in periodontal disease markers. Clin Chim Acta
2004;343(12):116.
60. Rai B, Kaur J, Jain R, Anand SC. Levels of gingival crevicular metalloproteinases8 and -9 in periodontitis. Saudi Dent J 2010;22(3):12931.
61. Last KS, Stanbury JB, Embery G. Glycosaminoglycans in human gingival
crevicular fluid as indicators of active periodontal disease. Arch Oral Biol
1985;30(3):27581.
62. Rossomando EF, Kennedy JE, Hadjimichael J. Tumour necrosis factor alpha in
gingival crevicular fluid as a possible indicator of periodontal disease in
humans. Arch Oral Biol 1990;35(6):4314.
63. Totan A, Greabu M, Totan C, Spinu T. Salivary aspartate aminotransferase,
alanine aminotransferase and alkaline phosphatase: possible markers in
periodontal diseases? Clin Chem Lab Med 2006;44(5):6125.

577

64. Ghallab N, Shaker O. Salivary-soluble CD44 levels in smokers and non-smokers


with chronic periodontitis: a pilot study. J Periodont 2010;81(5):7107.
65. Kojima T, Andersen E, Sanchez JC, Wilkins MR, Hochstrasser DF, Pralong WF,
et al. Human gingival crevicular fluid contains MRP8 (S100A8) and MRP14
(S100A9), two calcium-binding proteins of the S100 family. J Dent Res
2000;79(2):7407.
66. Cooke MS, Singh R, Hall GK, Mistry V, Duarte TL, Farmer PB, et al. Evaluation of
enzyme-linked immunosorbent assay and liquid chromatography-tandem
mass spectrometry methodology for the analysis of 8-oxo-7,8-dihydro-20 deoxyguanosine in saliva and urine. Free Radical Bio Med 2006;41(12):182936.
67. Zappacosta B, Manni A, Persichilli S, Boari A, Scribano D, Minucci A, et al.
Salivary thiols and enzyme markers of cell damage in periodontal disease. Clin
Biochem 2007;40(910):6615.
68. Ferrando R, Szponar B, Snchez A, Larsson L, Valero-Guilln PL. 3-Hydroxy fatty
acids in saliva as diagnostic markers in chronic periodontitis. J Microbiol Meth
2005;62(3):28591.
69. BaltacIoglu E, AkalIn FA, Alver A, Deger O, Karabulut E. Protein carbonyl levels
in serum and gingival crevicular fluid in patients with chronic periodontitis.
Arch Oral Biol 2008;53(8):71622.
70. Zhang L, Xiao H, Karlan S, Zhou H, Gross J, Elashoff D, et al. Discovery and
preclinical validation of salivary transcriptomic and proteomic biomarkers for
the non-invasive detection of breast cancer. PLoS One 2010;5(12):e15573.
71. Read GF, Wilson DW, Campbell FC, Holliday HW, Blamey RW, Griffiths K.
Salivary cortisol and dehydroepiandrosterone sulphate levels in
postmenopausal women with primary breast cancer. J Cancer Clin Oncol
1983;19(4):47783.
72. Baldini C, Giusti L, Bazzichi L, Lucacchini A, Bombardieri S. Proteomic analysis
of the saliva: a clue for understanding primary from secondary Sjgrens
syndrome? Autoimmun Rev 2008;7(3):18591.
73. Tishler M, Yaron I, Shirazi I, Levartovsky D, Yaron M. Salivary and serum soluble
interleukin-2 receptor in primary Sjogrens syndrome. Arch Oral Biol
1999;44(4):3058.
74. Higashi K, Yoshida M, Igarashi A, Ito K, Wada Y, Murakami S, et al. Intense
correlation between protein-conjugated acrolein and primary Sjogrens
syndrome. Clin Chim Acta 2010;411(56):35963.
75. Sfriso P, Ostuni P, Botsios C, Andretta M, Oliviero F, Punzi L, et al. Serum and
salivary neopterin and interferon-gamma in primary Sjogrens syndrome
Correlation with clinical, laboratory and histopathologic features. Scand J
Rheumatol 2003;32(2):748.
76. Zheng L, Zhang Z, Yu C, Tu L, Zhong L, Yang C. Association between IFN-[alpha]
and primary Sjogrens syndrome. Oral Surg Oral Med Oral Pathol Oral Radiol
Endod 2009;107(1):e128.
77. Jimnez-Alonso J, Sabio JM, Rivera-Cvico F, Martn-Armada M, Rodrguez M,
Jimez L, et al. Salivary and serum [beta]2-microglobulin and gammaglutamyl-transferase in patients with primary Sjgren syndrome and Sjgren
syndrome secondary to systemic lupus erythematosus. Clin Chim Acta
2003;334(12):22531.

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