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Nước bọt và protein trong ung thư miệng

Ind J Clin Biochem (Oct-Dec 2011) 26(4):326–334
DOI 10.1007/s12291-011-0149-8

REVIEW ARTICLE

A Review on Salivary Genomics and Proteomics Biomarkers
in Oral Cancer
Franky D. Shah • Rasheedunnisa Begum • Bhairavi N. Vajaria • Kinjal R. Patel
Jayendra B. Patel • Shilin N. Shukla • Prabhudas S. Patel



Received: 4 July 2011 / Accepted: 4 July 2011 / Published online: 9 August 2011
Ó Association of Clinical Biochemists of India 2011

Abstract Oral cancer has emerged as an alarming public
health problem with increasing incidence and mortality
rates all over the world. Therefore, the implementation of
newer screening and early detection approaches are of
utmost importance which could reduce the morbidity
and mortality associated with this disease. Sensitive and

specific biomarkers for oral cancer are likely to be most
effective for screening, diagnosis, staging and follow-up
for this dreaded malignancy. Unlike other deep cancers,
oral cancer is located in oral cavity. Hence, the direct
contact between saliva and oral cancer lesion makes the
measurement of tumor markers in saliva an attractive
alternative to serum and tissue testing. The DNA, RNA and
protein molecules derived from the living cancer cells can
be conveniently obtained from saliva. Thus, salivary biomarkers, a non-invasive alternative to serum and tissuebased biomarkers may be an effective modality for early
diagnosis, prognostication and monitoring post therapy
status. In the current post-genomic era, various technologies provide opportunities for high-throughput approaches
to genomics and proteomics; which have been used to
evaluate altered expressions of gene and protein targets
in saliva of oral cancer patients. The emerging field of
F. D. Shah Á B. N. Vajaria Á K. R. Patel Á
J. B. Patel Á P. S. Patel (&)
Biochemistry Research Division, The Gujarat Cancer &
Research Institute, Asarwa, Ahmedabad 380 016, Gujarat, India
e-mail: prabhudas_p@hotmail.com
R. Begum
Biochemistry Department, M.S. University of Baroda, Vadodara,
Gujarat, India
S. N. Shukla
The Gujarat Cancer & Research Institute, Asarwa, Ahmedabad
380 016, Gujarat, India

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salivary biomarkers has great potentials to prove its clinical
significance to combat oral cancer. Hence, we have
reviewed importance of several salivary genomics and
proteomics biomarkers for oral cancer.
Keywords Oral cancer Á Salivary biomarkers Á
Proteomics Á Genomics

Oral Cancer: A Leading Malignancy in India
Oral cancer is the 15th most prevalent cancer with the age
standardized incidence rate of 3.9 per 100,000 population
worldwide [1]. This dreaded malignancy stems as the
major health concern due to rising trends in younger population. The Indian subcontinent accounts for one-third of

the world burden of this malignancy [2]. In India, the age
standardized incidence rate of oral cancer is 12.6 per
100,000 population and a sharp increase in the incidence
rate of this cancer has been reported in recent years [3]. It is
the most common form of cancer and accounts for
increasing number of cancer related deaths among men in
India [2]. According to the rural and urban registry reports
of the Gujarat Cancer and Research Institute, Ahmedabad,
the estimated age standardized incidence rate of oral cancer
is 24 and 33.3 per 100,000 population, respectively [1]. A
recent pooled analysis from the International Head and
Neck Cancer Epidemiology consortium based on over
10,000 cases and 15,000 controls support significant role of
tobacco and alcohol use in etiology of oral cancer [4]. The
high incidence of oral cancer in India has also been linked
with habits of tobacco chewing and smoking [5]. The
repeated exposures of carcinogenic insults (e.g. tobacco
chewing) to oral mucosal cells increase the risk for
development of multiple independent premalignant and


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Fig. 1 Stages of oral cancer progression

malignant lesions from the accumulation of genetic alterations of oncogenes and tumor suppressor genes supporting
the ‘‘field cancerization’’ theory. In the field cancerization
model, multiple oral cancers develop from separate, independent cell clones [6].

Oral Cancer: Pathogenesis and Challenges
Oral cancer arises through a series of histopathologic
stages from benign hyperplasia to dysplasia to carcinoma
in situ followed by invasive squamous cell carcinoma
(Fig. 1). The malignancy is usually preceded by premalignant lesions like leukoplakia, erythroplakia and oral
submucous fibrosis with a transformation rate ranging from
0 to 20% in 1–30 years, according to the type of lesion. In
India, oral leukoplakia is considered to be potentially
malignant [7]. Globally, the 5 year mortality rate of oral
cancer is about 50% and has not changed significantly in
recent years despite of the advances in surgery, radiotherapy and chemotherapy. This is attributed mainly to late
diagnosis, poor response of tumor to chemotherapy and
radiotherapy as well as insufficient biomarkers for early
diagnosis and post therapeutic monitoring [8, 9]. The main
reason for late stage presentation of the disease is the
ignorance of lesions either by patients or clinicians. This is
also accounted due to lack of awareness of malignant
potentials of small lesions of oral cancer. Health education
programs aimed at motivating patients for early diagnosis
have also been largely unsuccessful because of incomplete
understanding of the disease [10]. Further, detection of an
oral cancer at stage I carries a prognosis of 80% survival,
while the same lesion at stage III carries a 20% survival.
This difference could affect not only the quality of life for

the patients but also the cost of the medical treatments of a
stage I versus stage III oral cancer patients. In addition,
early detection of cancer would also lead to fewer side
effects from cancer treatments such as chemotherapy and
radiotherapy and to a better prognosis. Moreover, oral
cancer has a very high recurrence rate. Patients who survive a first encounter with this disease have up to a 20 fold
increased risk of developing a second cancer [11, 12].
Thus, early identification of recurrence or a second primary
tumor remains an important challenge. Therefore, implementation of an early detection scheme would have a
positive impact on prognosis of the disease.
Microscopic investigations of the progressive cancer are
often conducted too late for intervention. It is also
impractical to use imaging techniques for cancer screening,
since they are time consuming and expensive. These
techniques are typically used for confirmation due to their
insensitivity for small lesions. Currently, the therapeutic
modalities for oral cancer patients are based on traditional
stage predicting indices and on histological grading.
However, these predictors are subjective and relatively
unreliable due to the nature of tumor and its response to
therapy [13]. Therefore, scientists have been searching for
alternative approaches, which can be helpful in early
diagnosis and ultimately improve mortality of oral cancer.
Moreover, better understanding of the biological nature of
this aggressive disease is also mandatory. There has been
ever growing efforts dedicated to the understanding of
basic biology of the disease. These efforts have been
focused on the identification of biological indicators for
early detection of its molecular nature and aggressiveness.
Recent advancements in oral cancer research have lead to
the development of potentially useful diagnostic tools at
clinical and molecular levels for early detection and better

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management of oral cancer. Latest tools developed for
early clinical detection of oral cancer include tolonium
chloride or toluidine blue dye, Oral brush biopsy kits,
ViziLite Plus, salivary diagnostics and several imaging
devices such as Velscope and multispectral optical imaging
systems [10]. However, oral cancer has remained a great
challenge and despite the wide availability of all advances,
no noticeable progress has been made in achieving earlier
diagnosis of this disease. One way of increasing the range
of diagnostic options in the case of primary oral tumors and
recurrence is to monitor the level of circulating tumor
markers which have adequate sensitivity and specificity.
Saliva has emerging role for the investigation of such circulating biomarkers which have relatively better sensitivity
and specificity with regards to diagnosis, prognostication
and treatment monitoring of the disease.

Saliva: The Mirror of Human Health
Saliva is a complex fluid composed of secretions from the
salivary glands and gingival crevicular fluid. Ninety percent of saliva is produced by the major salivary glands: the
parotid, submandibular and sublingual glands. Approximately, 10% of saliva is produced by minor salivary glands
clustered in the oral mucosa (lingual, labial, buccal, palatine, glossopalatine) [14]. Saliva has wide range of functions in normal human physiology (Fig. 2). It plays
important role in maintaining oral and dental health. It
participates in smooth ingestion and digestion of food;
mediate taste sensations. Saliva also exerts a wide range of
protective functions on oral tissues and teeth, including
facilitating the demineralization and remineralization of
teeth, modulating the adhesion of microorganisms to teeth
and other oral surfaces and buffering dietary acids [9, 15].
Saliva is an important body fluid for the detection of

Fig. 2 Functions and clinical utility of saliva

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physiological and pathological conditions of human body.
It is a complex and dynamic biological fluid containing
wide range of compounds. In addition, saliva is a good
indicator of the plasma/serum levels of various substances
such as hormones and drugs. In the last few years, scientific
interest has been raised to salivary analysis not only for
various compounds present into saliva (e.g., drugs, pollutants, hormones), but also for its well-documented relation
with bacterial, viral and systemic diseases. Therefore, it
can be used as a non-invasive method for monitoring
plasma concentrations of medicines or other substances
and for assessment of the severity of an illness [16].
Due to its diverse biological functions, salivary testing is
rapidly growing as a practical and reliable means in clinics
and research to recognize early signs of systemic illness
and exposure to risk factors. The components of saliva act
as a ‘‘mirror of the body’s health’’. Because of the widespread use and growing acceptability of saliva as a diagnostic tool, Various investigators have focused their efforts
to establish its clinical usefulness. The data have proved
significant utility for researchers, health care professionals
and community health program personals to detect and
monitor diseases and to improve general health of the
public.

Role of Salivary Biomarkers in Cancer
Till date, most of the biomarkers have been identified from
various body fluids. Among which blood and saliva are the
most widely studied body fluids that may contain reliable
biomarkers for detecting cancer. Saliva has the advantages
that it contains low background of normal material and
inhibitory substances as well as fewer complexes than
blood [17]. It is an informative body fluid containing an
array of analytes (Protein, mRNA and DNA) that can be
used as biomarkers for translation and clinical applications
[18]. Saliva has many advantages as a clinical tool over
serum and tissues, including simplicity of collection, storing and shipping, cost effectiveness, easy availability of
large sample volume for analysis and repeated sampling for
monitoring over time. The non-invasive saliva collection
techniques dramatically reduce anxiety and discomfort of
the patients. Saliva is also easier to handle for diagnostic
procedures because no special equipment is needed for
saliva sample collection and it does not clot, thus reducing
the manipulations which may be required for biochemical
analysis [19].
Clinical significance of salivary biomarkers in various
malignancies is studied by several investigators. Streckfus
et al. [20, 21] explored for the presence of salivary proteomics and genomics signatures for breast cancer. The
authors reported Her2/neu as the first salivary biomarker


Ind J Clin Biochem (Oct-Dec 2011) 26(4):326–334

for breast cancer and also documented raised levels of CA
15-3 and Her2/neu as well as low levels of p53 in patients
with breast cancer. Chen et al. [22] described elevated
salivary levels of CA 125 in patients with ovarian cancer.
Schapher et al. [23] also suggested that salivary leptin was
expressed in much higher amount in salivary gland tumors
than in healthy parotid tissue. It has been reported that
gastric cancer can also be identified at an early stage by
using saliva proteome analysis [24]. Wong et al. [25] have
also identified that combination of three mRNA biomarkers
(acrosomal vesicle protein 1, ACRV1; DMX like 2,
DMXL2 and dolichyl phosphate mannosyltransferase
polypeptide 1, catalytic subunit, DPM1) could differentiate
pancreatic cancer patients from chronic pancreatitis and
healthy individuals.
Thus, the saliva based analysis; a non-invasive alternative to serum analysis can be an effective modality for
diagnosis and prognostication of cancer as well as for
monitoring post-treatment therapeutic response of the
patients. Hence, the development of salivary diagnostic
tools is of paramount importance, especially in identification of high risk group, patients with premalignant lesions
and patients with previous history of cancer [26].

Salivary Genomics and Proteomics Biomarkers in Oral
Cancer
Among all the malignancies, oral cancer is one such
malignancy where saliva examination for detection can
show the greatest benefit because of its direct contact with
oral cancer lesions. The most important point for selecting
saliva as a diagnostic tool is that it also contains the fallen
cells in oral cavity which allow saliva to be the first choice
of screening and identification for potentials biomarkers for
oral cancer [8]. Several reports on salivary biomarkers in
oral cancer have shown significant clinical usefulness for
oral cancer as summarized in Table 1. As pointed out in the
table, earlier reports have focused on salivary ‘‘tools’’ for
measuring changes in specific salivary molecules such as
proteins or nucleic acids. The studies have examined
genomic and proteomic targets such as DNA aberrations,
mRNAs, enzymes, cytokines, growth factors, metalloproteinases, telomerase, cytokeratins etc. in oral cancer [13,
17, 27–31]. The first report of saliva as a diagnostic tool for
oral cancer detection was published in 2000 by Liao et al.
[32]. The authors claimed that exon 4, codon 63 of the p53
gene was mutated in salivary DNA from five of eight
(62.5%) oral cancers patients. In addition, autoantibodies
against p53, the aberrantly expressed protein in patients
with oral cancer has been identified in both saliva as well as
serum [33–35]. TP53 was the only gene with similar
incidences of loss of heterozygosity (LOH) and mutations.

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LOH was shown to occur more frequently than gene
mutations in oral cancer. El-Naggar et al. [36] found that
49% of the saliva samples of oral cancer patients had LOH
in at least one of the 25 markers studied.
It is well established that oxidative stress plays an
important role in progression of oral cancer. Bahar et al.
[37] documented that salivary reactive nitrogen species
were significantly higher, while all salivary antioxidants
were significantly lower in the oral cancer patients as
compared to the controls. This increase in reactive nitrogen
species may be the event leading to the consumption and
reduction of salivary antioxidants resulting in the oxidative
damage to DNA and proteins, and possibly leading to
progression of oral cancer. Recently, Shiptzer et al. [13]
reported increased salivary levels of cell cycle regulatory
proteins including Cyclin D1 and ki67, glycolytic enzyme
lactate dehydrogenase (LDH), matrix metalloproteinase
(MMP)-9, as well as reduction in DNA repair enzyme,
8-oxoquanine DNA glycosylase (OGG1) and Maspin, a
tumor suppressor protein in oral cancer patients. Sato et al.
[38] found significantly increased interleukin (IL)-6 levels
in saliva of oral cancer patients than controls. Brailo et al.
[39] also studied alterations in salivary IL-6 and tumor
necrosis factor alpha (TNF-a) in patients with oral leukoplakia. They observed that salivary IL-6 and TNF-a levels
were significantly higher in patients with oral leukoplakia
as compared to the healthy individuals. IL-6 inactivates
p53 tumor suppressor gene by supporting the hypermethylation of its promoter region which results in suppression of apoptosis and uncontrolled cell proliferation. TNFa activates NFjB transcription factor which stimulates cell
proliferation and blocks apoptosis and additionally enhances secretion of proinflammatory cytokines. Rhodus et al.
[27] reported significantly higher salivary levels of IL-1,
IL-6, IL-8 and TNF-a in oral cancer patients as compared
to the patients with dysplastic oral lesions and controls.
Considering, the fact that same cytokines were significantly
elevated in both oral cancer and oral premalignant lesions,
it may have a diagnostic value as the marker of malignant
transformation of oral premalignant lesions. In addition,
IL-6 also correlated with the recurrence of oral cancer [40].
Zhong et al. [30] found 75% positive expression of
telomerase in saliva of oral cancer patients suggesting its
usefulness as a supportive marker to diagnose oral cancer
and also suggested that human telomerase reverse transcriptase (hTERT) analysis may be a potential biomarker for
the diagnosis of oral cancer. Telomerase is a ribonucleoprotein which aid to elongate repeat sequence at the end of
the chromosomes. Telomerase reactivation might be prerequisite for development of malignant cells from the
somatic cells by escaping from the proliferative limitations
of cellular senescence. Recent research has been directed
towards detecting the human papilloma virus (HPV) in

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Table 1 Clinical significance of various salivary biomarkers in oral cancer
Biomarkers

Inference

Ref

CycD1, Ki67

All the biomarkers were significantly altered in oral cancer and found to be useful as a supportive tool for
diagnosis, prognosis and post-operative monitoring.

[13]

These proangiogenic, proinflammatory cytokines were found to be elevated in whole saliva of oral cancer
patients and oral precancers as compared to controls which suggested its utility as surrogate indicators of
carcinogenic transformation from oral precancer to oral cancer.

[27]

Telomerase

Increased telomerase activity found in saliva of oral cancer patients suggested that the telomerase in saliva
could be useful biomarker for oral cancer.

[30]

p53 Autoantibodies

Presence of p53 autoantibodies in saliva as well as serum of oral cancer patients demonstrated that its
detection in saliva can offer a non-invasive method for the detection of a subset of tumors with p53
aberrations.
Altered salivary levels of reactive nitrogen species and antioxidants in oral cancer patients suggested the
possibility of a direct link between salivary free radicals, antioxidants and oral cancer which may
contribute to its diagnosis and treatment.

[33]

LDH, MMP-9
OGG1, Maspin
IL-1, IL-6
IL-8, TNF-a

Reactive nitrogen
species

[37]

IL-6, TNF-a

Significantly higher levels of salivary IL-6 and TNF-a were observed in patients with oral leukoplakia
compared to healthy controls. The alterations in salivary IL-6 and TNF-a might play a significant role in
development of oral leukoplakia.

[39]

HPV

Detection of HPV in salivary rinses has potentials for development of molecular screening for HPV-related
oral cancer.

[41]

M2BP, MRP14

The data proved that these new targets may lead to a simple clinical tool for the non-invasive diagnosis of
oral cancer and suggested that patient-based salivary proteomics is a promising approach to the discovery
of biomarkers for oral cancer detection.

[42]

Actin, myosin

Actin and myosin are promising salivary biomarkers for distinguishing premalignant and malignant oral
lesions.

[43]

Transferrin

Salivary transferrin levels in oral cancer patients strongly correlated with the size and stage of the tumor.

[44]

Salivary mRNA

The study developed a method for the multiplex RT-PCR, which made it possible to examine a large
number of mRNAs from one droplet of saliva.

[45]

DNA hypermethylation

Methylation array analysis of saliva can produce a set of cancer related genes that are specific and can be
used as combined biomarkers for early detection of oral cancer. An assay was developed that could
rapidly quantify the promoter hypermethylation of the gene of interest and could potentially be applied
into a clinical setting.

[46, 48]

Salivary IL-8 mRNA
and protein

EC sensor was developed with multiplexing biomarker detection for salivary diagnostics. IL-8 mRNA and
IL-8 protein levels measured by the EC sensors showed significant differences between oral cancer
patients and controls.

[49]

CD59, Profilin 1
Catalase

saliva, as one of the etiological factor in oral cancer. The
incidence of HPV positivity in patients treated for oral
cancer is estimated to be more than 45% [9, 41].
The cellular and molecular heterogeneity of oral cancer
and the large number of genes potentially involved in oral
carcinogenesis emphasize the importance of studying gene
expression changes in a global scale by proteomics. The
modern high throughput genomic and proteomic approaches have been extensively used to study the altered
expressions of genes and proteins in oral cancer. It may be
helpful to facilitate the identification of potential biomarkers for oral cancer. With the advances in mass spectrophotometry, there is ongoing development in salivary
proteomics for biomarker identification of oral cancer. Indepth analysis of human salivary proteome by Hu et al.
[42] revealed several salivary proteins at differential levels
between oral cancer patients and matched controls. The

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approach successfully validated five candidate biomarkers
including Mac-2 binding protein (M2BP), myeloid related
protein 14 (MRP14), CD59, profilin 1 and catalase using
immunoassays on independent set of oral cancer patients
and matched controls [39]. Using quantitative proteomics
approach, de Jong et al. [43] also observed consistently
increased levels of actin and myosin in saliva samples from
individuals with malignant oral lesions as compared to the
premalignant lesions. The authors concluded that actin and
myosin are promising salivary biomarkers for distinguishing premalignant and malignant oral lesions. Salivary
transferrin is also validated as a biomarker for detection of
early stage oral cancer [44]. The tumor-specific DNA in
saliva could also be used as biomarker for oral cancer [45].
Methylation array of salivary DNA was supported as an
effective biomarker for early detection of oral cancer [46].
Hypermethylation on the promoter of DNA in specific gene


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such as p16 was found in serum as well as also in saliva
[47, 48]. Since mRNA is the direct precursor of protein and
as the corresponding levels are correlated in cells and tissue
samples, salivary mRNA for dual specificity phosphatase 1
(DUSP1), H3 histone, family 3A (H3F3A), IL-1B, IL-8,
ornithine decarboxylase antizyme 1 (OAZ1), spermidine/
spermine N1-acetyltransferase (SAT) and S100 calcium
binding protein P (S100P) are documented as the biomarkers of oral cancer [45, 49]. Brinkmann et al. [50] also
validated four transcriptome [IL-8, IL-1B, spermidine/
spermine N1-acetyltransferase 1 (SAT1) and S100P] and
three protein (IL-1B, IL-8 and M2BP) biomarkers which
were significantly elevated in oral cancer. The authors also
concluded that these biomarkers are discriminatory and
reproducible in different ethnic cohorts. Several studies on
polymorphism of several genes like IL-6, IL-8, TNF-a,
vascular endothelial growth factor (VEGF), cytochrome
P4501A1 (CYP1A1), glutathione-S-transferase T1 (GSTT1)
and glutathione-S-transferase M1 (GSTM1) were found to
be associated with the development of oral cancer [51–55].
Saliva is a convenient source of genomic DNA that has
been proposed to offer non-invasive approach than blood/
tissue based analysis. Thus, it can be envisioned that salivary genomics will eventually be used for genomic
screening of oral cancer. Hence, further studies with large
cohort size are required to evaluate the role of gene polymorphism in oral cancer development from the fallen
cancer cells in saliva.

Recent Data on Salivary Biomarkers from Our
Laboratory
We have studied several biomarkers associated with cancer
susceptibility invasion and metastasis as well as glycosylation from saliva samples obtained from 53 oral cancer
patients and 53 controls.
Susceptibility Markers in Saliva
We evaluated role of CYP1A1, GSTT1 and GSTM1 gene
polymorphism from saliva to assess their role as cancer
susceptibility markers. The CYP1A1 gene encodes an
enzyme with aryl hydrocarbon hydroxylase activity. Formation of aryl epoxides by aryl hydrocarbon hydroxylase is
the first step in the metabolism of polycyclic aromatic
hydrocarbons from tobacco [53]. GST, a very important
Phase II family of enzymes catalyzes the detoxification of a
wide variety of active metabolites of tobacco carcinogens.
GSTM1 catalyzes the conjugation of glutathione tripeptide
to polycyclic aromatic hydrocarbons diol epoxides whereas
GSTT1 participates in detoxification of monohalomethanes
and reactive diol epoxides [54, 55]. Polymorphisms in the

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genes that code for these enzymes that are potentially
involved in either the activation (Phase I) or detoxification
(Phase II) of chemical carcinogens in tobacco may alter
expression or function, thus increasing or decreasing the
activation or detoxification of carcinogenic compounds.
The CYP1A1 gene polymorphism (CYP1A1*2A) in our
study showed that the prevalence of CYP1A1 homozygous
wild (m1/m1) genotype was 56% in cases and 48% in
controls. The distribution of CYP1A1 heterozygous (m1/
m2) genotype was 32% in cases and 48% in controls.
Whereas, the prevalence of CYP1A1 homozygous variant
(m2/m2) genotype was 12% in cases and 8% in controls.
GSTM1 analysis from saliva showed that 36% of controls
and 36% of patients represent GSTM1 null genotype which
is in accordance with our previous blood based studies
[56]. Salivary analysis of GSTT1 showed that the frequency
of GSTT1 null genotype was 24% in oral cancer patients
and 16% in controls. Null GSTT1 genotype was found to be
higher in oral cancer patients as compared to the controls
which suggested that individuals with null genotype may
be more susceptible to develop oral cancer (Unpublished
data).
Invasion and Metastasis Related Salivary Biomarkers
It is well documented that oral cancer has a high degree of
local invasiveness and a high rate of metastasis to cervical
lymph-nodes. Tissue invasion and metastasis requires
extensive remodeling and degradation of extra cellular
matrix and basement membrane. Gelatinases; especially,
gelatinase A i.e. MMP-2 and gelatinase B i.e. MMP-9 have
been shown to play a significant role in invasion and
metastasis as they degrade type IV collagen, a major
component of basement membrane. Our results indicated
that salivary levels of MMP-2 and MMP-9, the invasion
and metastasis related markers also play important role in
the pathogenesis of oral cancer (Unpublished data). We
observed that both active as well as latent forms of salivary
MMP-2 and MMP-9 were significantly higher in oral
cancer patients as compared to the controls. The receiver
operating characteristic (ROC) curve analysis revealed that
all forms of MMP-2 and MMP-9 could significantly discriminate between oral cancer patients and controls. The
results are in accordance with our previous study on MMP2 and MMP-9 from plasma and tissues obtained from oral
cancer patients [57–59].
Salivary Glycoproteins
Aberrant glycosylation is the universal feature of cancer.
Various glycoconjugates are released into circulation
through increased turnover, secretion and/or shedding from
malignant cells. Earlier reports from our laboratory

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documented elevated serum levels of total sialic acid
(TSA), lipid bound sialic acid (LSA), and TSA to total
protein ratio in oral cancer and also suggested that alterations in sialic acid levels have significant clinical usefulness for oral cancer patients [60–63]. Our recent data on
salivary glycoproteins including TSA and a-L-fucosidase
showed that the changes in these biomarkers play an
important role in oral carcinogenesis. It was observed that
salivary a-L-fucosidase levels were significantly higher in
oral cancer patients as compared to the controls. Also, the
TSA/total protein ratio was found to be significantly
increased in oral cancer patients as compared to the controls. ROC curve analysis revealed that a-L-fucosidase
levels have significant efficacy to discriminate between
oral cancer patients and controls (Unpublished data).
Electrochemical Sensor for Multiplex Salivary
Biomarker Detection
In a collaborative research project with University of
California at Los Angeles (UCLA), Los Angeles,
California, USA, we developed an electrochemical (EC)
sensor based on the simultaneous detection of multiple
salivary biomarkers for oral cancer. We have found that
multiplex assay of both IL-8 mRNA and IL-8 protein levels
measured by the EC sensors have significant differences
between oral cancer patients and controls. Under the multiplexing mode, the limit of detection of salivary IL-8
mRNA reaches to 3.9 fM and 7.4 pg/ml for salivary IL-8
protein. The ROC analysis revealed that the EC sensor
yields as high as 90% sensitivity and specificity for both IL8 mRNA and IL-8 protein. These results were very close to
the data measured by traditional assays (enzyme-linked
immunosorbent assay, ELISA and polymerase chain reaction, PCR) using same saliva samples. The area under curve
(AUC) value was 0.90 for IL-8 mRNA and 0.91 for IL-8
protein. While the combined IL-8 mRNA and protein
showed better AUC (0.93) compared with single biomarker
[49]. Therefore, combination of multiple biomarkers
exhibited improved accuracy instead of single biomarker
for oral cancer detection. Promising results from these data
suggested that in-depth analysis of these salivary biomarkers have a great potentials to be clinically useful noninvasive biomarkers for screening, diagnosis and treatment
monitoring of oral cancer patients.

Concluding Remarks
Currently, the saliva research field is rapidly evolving and
advancing due to the use of novel approaches including
metabolomics, genomics, proteomics and bioinformatics.
Implication of saliva as a diagnostic tool for various

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Ind J Clin Biochem (Oct-Dec 2011) 26(4):326–334

diseases has proved that saliva contains much more clinical
information apart from its functional value. Due to its
proximity to oral cavity and non-invasive collection procedure, salivary screening can be the best choice as primary screening test for oral cancer. The systematic analysis
of salivary genomics and proteomics biomarkers facilitates
the identification of sensitive and specific parameters for
oral cancer that may aid in effective screening to identify
patients with high risk and also help in designing better
treatment modalities thus improving survival of oral cancer
patients. Collectively, the promising field of salivary
genomics and proteomics biomarker analysis may
strengthen and transform the field of oral cancer diagnosis
which will enable clinicians to monitor patients’ saliva for
diagnosis and prognostication of oral cancer. It will thus
advance the clinical efforts to overcome the severity of the
disease. However, there may be cultural and behavioral
perceptions against using saliva; these barriers will need to
be overcome with time. Further, enormous efforts from
researchers and clinicians are essential to turn salivary
diagnostics into clinical and commercial reality to combat
oral cancer.
Acknowledgment Our ongoing work on salivary biomarkers is
partially funded by research project grants from Indian Council of
Medical Research (ICMR, Grant no.: 5/13/95/2003-NCD III) and
Gujarat Cancer Society, Ahmedabad (Grant no.: RE/28/BRD 1/09).
The authors are also thankful to The Gujarat Cancer and Research
Institute for administrative support and allowing to use the clinical
material.

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