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Oral Oncology
journal homepage: www.elsevier.com/locate/oraloncology
Review
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
570
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
571
(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.
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
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
572
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
Cask-interzcting protein 2
Actin-related protein 5
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
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
Putative S100
calcium-binding
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
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
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
574
Table 5
Potential biomarkers for breast cancer detection.70
Blood biomarkers in saliva
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)
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
575
Lactoferrin b-2microglobulin
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
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
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.
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