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Review

DOI: 10.5582/ddt.2015.01042

Oesophageal squamous cell carcinoma (ESCC): Advances

through omics technologies, towards ESCC salivaomics
Juan Jos Gonzlez-Plaza1,2,*, Nataa Hulak3, Eduardo Garca Fuentes4,5, Lourdes Garrido-Snchez5,6,
Zhaxybay Zhumadilov1, Ainur Akilzhanova1
1

Laboratory of Genomic and Personalized Medicine, Center for Life Sciences, PI National Laboratory Astana, AOE Nazarbayev
University, Astana, Kazakhstan;
2
Research Department, University Hospital for Infectious Diseases "Dr. Fran Mihaljevi", Zagreb, Croatia;
3
Department of Microbiology, Faculty of Agriculture, University of Zagreb, Zagreb, Croatia;
4
Unidad de Gestin Clnica de Endocrinologay Nutricin, Instituto de Investigacin Biomdica de Mlaga (IBIMA), Hospital
Regional Universitario, Mlaga, Spain;
5
CIBER Fisiologa de la Obesidad y Nutricin (CIBEROBN), Mlaga, Spain;
6
Unidad de Gestin Clnica de Endocrinologa y Nutricin, Instituto de Investigacin Biomdica de Mlaga (IBIMA), Hospital
Clnico Virgen de la Victoria, Mlaga, Spain.

Summary

Oesophageal Squamous Cell Carcinoma (ESCC) is one of the two main subtypes of
oesophageal cancer, affecting mainly populations in Asia. Though there have been great
efforts to develop methods for a better prognosis, there is still a limitation in the staging of
this affection. As a result, ESCC is detected at advances stages, when the interventions on the
patient do not have such a positive outcome, leading in many cases to recurrence and to a very
low 5-year survival rate, causing high mortality. A way to decrease the number of deaths is
the use of biomarkers that can trace the advance of the disease at early stages, when surgical
or chemotherapeutic methodologies would have a greater effect on the evolution of the
subject. The new high throughput omics technologies offer an unprecedented chance to screen
for thousands of molecules at the same time, from which a new set of biomarkers could be
developed. One of the most convenient types of samples is saliva, an accessible body fluid that
has the advantage of being non-invasive for the patient, being easy to store or to process. This
review will focus on the current status of the new omics technologies regarding salivaomics in
ESCC, or when not evaluated yet, the achievements in related diseases.
Keywords: Oesophageal squamous cell carcinoma, saliva, salivaomics, transcriptomics,
proteomics, metabolomics

1. Introduction
Oesophageal Cancer (EC) has two main subtypes
with different pathological features, Adenocarcinoma
(EAC) and ESCC (1,2), representing between them
more than 90% of the detected cases (3). There is also
a different trend in the geographical distribution for
both EC subtypes, being that of EAC in the Western
world (4), while ESCC is especially present in Asia,
for example in China (5-7), Iran (8,9), Japan (10), or

*Address correspondence to:

Dr. Juan Jos Gonzlez Plaza, University Hospital for
Infectious Diseases "Dr. Fran Mihaljevi" Research Department
Mirogojska 8, 10 000 Zagreb, Croatia.
E-mail: [email protected]

Kazakhstan (11). In this last country, ESCC is the 6th

major type of diagnosed cancer, with high mortality
rates among women (9th place) and men (5th place) (11).
Early detection of ESCC and subsequent treatment
would be crucial in order to decrease mortality (12), but
as indicated by several authors (4,13-15) lack of early
stage diagnostic tools is one of the biggest problems
in ESCC diagnostics, especially because ESCC is
manifested as asymptomatic lesions at first stages.
Some of the current techniques used for diagnosis
are non-invasive imaging methods, as well as more
conventional ones that include computed tomography
(CT) scan, or endoscopic ultrasound (EUS). Some of
the difficulties that these techniques face are in the case
of EUS the limitation that tumour enlargement pose
for the passage of endoscope in advanced cases, or for

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CT the lower sensitivity displayed in comparison to a
combination of PET (Positron Emission Tomography)
and the use of 2-deoxy-2-( 18 F)fluoro-D-glucose
(18F-FDG) (16). As previously indicated by Takeshita
(15) there have been advances in the use of several
techniques as those mentioned above, but still a late
detection happens at very advanced stages (17,18),
when surgical interventions are not effective and lead to
recurrence and low survival rates (19).
Besides, an additional problem for ESCC is the
multifactorial nature of its occurrence (20), and the
influence that different habits would have over the risk
of developing this disease, as it has been associated with
heavy smoking, drinking, or low intake of vegetables
or fruits (21-23). The increased risk factor in this case
as indicated by Cheng and Day (22) may come as a
consequence from a direct contact of the potential
carcinogen with the epithelium, some existing transport
facilitating mechanism, or derived from compounds that
increase the cell turnover in the epithelial cells. Albeit yet
a controversial topic, there have been also studies trying
to correlate the occurrence of HPV infections and ESCC,
though results showed an elevated degree of variation
that did not yield a clear association between the viral
infection and the development of the disease (24-26).
A molecular feature that could be associated to the
development of this disease would serve as an indicator
for a more effective treatment. In this scenario,
the discovery of biomarkers would become a great
advantage for clinicians allowing an early diagnosis of
ESCC, as it has been for many other diseases, e.g. level
of serum creatinine as indicator of renal function. The
term biomarker (biological marker) can be defined as "a
characteristic that is objectively measured and evaluated
as an indicator of normal biological processes,
pathogenic processes, or pharmacologic responses to a
therapeutic intervention", definition that was proposed
at the 1998 National Institutes of Health Biomarkers
Definitions Working Group (27,28). Biomarkers can
aid in diagnosis, and an efficient way to discover
them is the use of available omics technologies.
Transcriptomics, Proteomics, or Metabolomics (TPM)
are relatively new high throughput techniques that allow
performing massive screenings of different molecules.
Each of them refer to the complete set of transcripts/
proteins/metabolites (respectively) and their quantity,
for a given cell with a given genotype, under certain
environmental conditions (including developmental or
physiological stage, as well as being under influence of
bioregulators) (29-32). After this definition, it comes
as a logic consequence that physiological changes
that accompany the development of a disease in
humans lead to changes in the TPM profile, that can
be observed and measured in a tissue, or indirectly
measured/observed in human fluids, e.g. urine, sweat,
saliva. Currently due to the development of omics
technologies, these profile changes can be detected

248

and analysed (29-32). The advantage of using TMP

"expression" biomarkers is that they are closely related
to the disease, as they are usually by-products of its
development. Metabolites are for example end-points
of all the system, being the final products of metabolic
pathways (33) and a reflection of the phenotype (34).
Therefore, this approach would yield biomarkers that
are potentially specific for the studied disease, and
have a potentially direct application (35). The use of a
combination of this new technologies (36), can largely
contribute in the understanding of each disease, their
evolution, and molecular mechanisms.
A very important point in studies concerning
cancer or other diseases, is the validation of the model
through testing in an independent set of samples (36).
This would be, for example, a set of individuals with
the same features as the control (healthy), and another
set of independent affected ones, which were not used
in the original screening. It will mean that the found
biomarkers have the power to potentially predict or
be associated with the disease in any given set of
samples that fulfil the disease conditions. Otherwise,
this would be (the necessary) technical validation of the
study. Biomarkers that are validated in this way can be
promising targets for further clinical testing assays.
Considering the final aim of developing biomarkers
to routinely use them for ESCC testing in clinical
settings, one of the most accessible and interesting
types of human samples is saliva (35). It has numerous
advantages, being easily accessible for clinicians, and
non-invasive for patients (37-39). Compared to imaging
techniques, saliva collection is methodologically less
demanding than a 18F-FDG PET assay (16), allowing to
scale up the number of patients to sample. Compared
with other identification techniques as EUS, its
advantage is that saliva collection avoids the anxiety
that an endoscopy may cause to the patient, or the
potential disturbances afterwards. Other interesting
features of saliva in comparison to blood, is that saliva
does not need dedicated precautions for storage or
biosecurity, besides it does not clot (14). Thus, its
handling is very convenient for any hospital facility
worldwide, especially in depressed or impoverished
areas.
Saliva is a human fluid that is originated mostly
in three salivary glands (parotis, submandibularis and
sublingualis), as well as a number of minor glands, and
the fluid from gingival crevice (37,40). It is a complex
mixture of exudates derived from mucosa, plasma,
microflora (or what we will refer as oral metabiome/
microbiome), epithelial cells, small metabolites and
different transcripts, among other minor compounds
(41-44). This complexity must be considered as a very
important feature for its study. Regarding the protein
content, some studies reported that approximately
20%-30% of the proteins that can be found in blood
plasma are present in saliva (45,46). According to

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Castagnola et al., most of the proteins (more than 90%

of representation) are not from gland secretory origin,
but common to other body fluids or tissues. However,
despite this abundance, common proteins account only
for 15% w/w of the salivary proteome, something
that has to be considered as a potential drawback for
biomarker discovery studies (41). An aspect of saliva
that cannot be forgotten, of importance because it can
affect the results, is the human oral microbiome (47,48).
It can be defined as the population of different bacterial
species that inhabit the oral cavity, while they form part
of the whole human microbiota. Taking in account all
of the above mentioned features from saliva, there is
room for discovery of different biomarkers associated
to the development of ESCC.
In conditions of disease, human saliva can reflect
the physiological state as occurs in blood (49,50).
This has been demonstrated in the work of Asatsuma
et al. (51) (Table 1), in which they found significant
differences in a protein between the healthy patients
compared with the ones affected by primary Sjgren's
syndrome (pSS). Nevertheless, this disease affects
salivary and lachrymal glands (52), and it could be
thought that these type of alterations are easier to trace
in saliva than other conditions, for which could not be
possible the discovery of useful biomarkers. Then it is
important to address here the important findings in a
disease that is not directly related with the oral cavity,
Breast Cancer (BC), where saliva was the sample of
choice. A successful example is the study of Zhang and
collaborators (53) (Table 1), where they discovered
and validated eight mRNA biomarkers and a protein
biomarker, having a 92% accuracy in the tested sample
set.
It is then clear that saliva diagnostics is a powerful
tool and at the same time a promising biofluid. The
current status regarding ESCC salivaomics will be
reviewed when available in the following sections for
the three main current high throughput technologies:
transcriptomics, proteomics, and metabolomics. For
clarity, the most promising results in salivary biomarker
discovery have been briefly resumed in Table 1.
2. Transcriptomics
Transcriptomics studies and quantifies the set of RNA
molecules produced by the genome as a result of the
environmental influences, or the developmental stage. A
major question that has to be addressed is the stability
of RNA in saliva. It is a common presumption that RNA
cannot be stable in saliva (54) due to its labile nature
and the presence of RNases in the oral cavity (55). A
further complication for cancer studies is the reported
higher activity of RNases in gastric cancer patients (56),
leading to a higher degradation of total RNA in the oral
cavity of affected subjects. If that described situation
applies for other types of cancer, being ESCC of our

interest, chances to find intact RNA are lower and thus

the capability to discovery useful potential biomarkers
will decrease vastly. There have been successful studies
focusing on saliva as the sample material for oral
squamous cell carcinoma (OSCC), for example the
work of Li and collaborators (55) (Table 1), where they
found more than 1,600 differentially expressed genes.
However some other studies (54) have described that
the observed signalling molecule was in fact DNA, not
RNA, what complicates the analysis. Notwithstanding,
Li and colleagues were the first ones to observe more
than 3,000 different RNA transcripts in human saliva
(44) (Table 1), according to what they report in a more
recent study from their laboratory, in which Park and
colleagues (57) (Table 1) further characterized the
stability of RNA in saliva. This latter study tested
whether informative RNA molecules exist in saliva
or not through different molecular biology assays. As
reported (57) , until 2004 most of the detected RNA
had a viral or bacterial origin. In this study Park and
colleagues (57) used a 22,283 cDNA probes microarray
(Agilent Affymetrix Human Genome U133A) to
test the complexity of several oral saliva samples in
terms of number of distinct transcripts that could be
detected, having found more than 6,000 in the whole
saliva. Regarding stability, one of the possibilities that
they indicate for the stability of RNA in saliva is the
association with mucin, protein that would protect from
degradation, as well as other type of macromolecules.
Are there additional sources of cell free circulating
RNA in saliva? Amidst the possible origins some
authors have indicated apoptotic processes (58), while
other studies report the presence of exosomes (59,60).
In keeping with this last possibility, Ogawa and
collaborators (59) (Table 1) found for the first time the
presence of exosomal vesicles in whole saliva samples
from humans.
For efficient biomarker discovery, the release of
RNA from the apoptotic cells has to occur in a quantity
that allows an early identification and efficient tracking
of the disease. The appearance of RNAs in latter stages
of the disease will not have such a clinical value for
ESCC, as there are other available techniques that
were mentioned in the previous section, and because
of the current critical need for early markers. In that
way, the presence of exosomes and their nature as a
communication mean between distant cell types (61-64),
can be a key point to exploit biomarker discovery. That
would be of a great interest in the case of ESCC, as the
chances for detecting apoptotic derived RNA may not
be so big during early stages than in advanced ones, but
still tumorigenic cells may be starting to derive RNA
containing exosomes for communication while the
lesion is not yet detectable.
More interesting developments related with saliva
that focused on studying BC and their potential salivary
biomarkers, were achieved by Zhang and collaborators

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Table 1. Most promising advances regarding biomarker discovery in saliva

Methodology

Model of study

ELISA; sandwich EIA

Discovery

Importance

Ref.

pSS

Significantly increased levels of

MMP-9/TIMP-1 and MMP-9 in pSS
patients

Use of saliva to study differences

between affected patients and healthy
ones

Asatsuma
et al. (51)

Affymetrix Human Genome

U133A
Array

Healthy subjects

3,000 different RNA transcripts in

human saliva

Large scale methodology to study

transcriptomics in saliva

Li
et al. (44)

Affymetrix Human Genome

U133A
Array

OSCC

1,600 differentially expressed genes

Differentially expressed genes between

affected patients and healthy ones
through large scale approach

Li
et al. (55)

Affymetrix microarray
platform/2D-DIGE

BC

Discovery and validation of eight

mRNA, and a protein biomarker
(92% accuracy)

Salivary biomarkers in a disease not

related with oral cavity, broadening
the field

Zhang
et al. (53)

Affymetrix Human Genome

U133A
Array

Healthy subjects

Characterization of RNA stability in

saliva

Observation of the association with

mucin and other macromolecules, for
protection against degradation

Park
et al. (57)

Peptide sequencing/MALDITOF-MS

Healthy subjects

Exosomes in saliva samples

First time report of exosomal vesicles

in whole saliva samples from humans

Ogawa
et al. (59)

miRNA microarray

EC

Different miRNA profiles in saliva

derived from healthy patients and
affected ones

Four validated miRNA biomarkers in

EC

Xie
et al. (70)

RNAseq

Healthy subjects

20-25% of sequenced reads that align

to the human genome, while another
30% aligns to HOMD

First whole RNAseq in saliva

samples from healthy human subjects,
methodology to differentiate microbiota
from human moiety

Spielmann
et al. (43)

RNAseq

Healthy subjects

Human oral and gut microbiome and

transcriptome differences

Establishment of patient self-collection

of samples

Franzosa
et al. (72)

RNAseq

PD

Oral metabiome differences between

healthy microbiome and disease
microbiome

Found differences in the diversity of

the community between disease and
healthy states

Jorth
et al. (73)

RNAseq

Healthy subjects

exRNAs as micro RNA, Piwiinteracting RNA, and circular RNA

Characterization of diverse RNA

species from saliva.

Bahn
et al. (74)

Peptide sequencing/MS

Healthy subjects

Characterization of the salivary

metaproteome

First catalogue of metaproteome,

serving as a reference for future studies

Jagtap
et al. (76)

Subtractive proteomics
approach, combination of
separation techniques: LC,
LC-MS/MS, QqTOF MS

OSCC

Differential levels of proteins

between healthy and affected patients

Verification of results in independent

set of patients and healthy subjects,
promising biomarkers

Hu
et al. (81)

CE-TOF-MS

BC, OC, PC, PD

57 metabolites for accurate prediction

of the probability of each disease

Shows the feasibility to obtain

valuable information and biomarkers
from saliva, in a variety of cancer
diseases

Sugimoto
et al. (85)

ELISA: enzyme-linked immunosorbent assay; EIA: enzyme immunoassay; pSS: primary Sjgren's syndrome; MMP-9: metalloproteinase-9 ;
TIMP-1: tissue inhibitor of metalloproteinase-1; 2D-DIGE: two-dimensional dierence gel electrophoresis; BC: Breast Cancer; OSCC: Oral
Squamous Cell Carcinoma; MALDI-TOF-MS: matrix-assisted laser desorption/ionization time-of-ight mass spectrometry; EC: Oesophageal
Cancer; PD: periodontal disease; HOMD: Human Oral Microbiome Database; exRNAs: extracellular RNAs; LC: liquid chromatography; MS:
mass spectrometry; QqTOF: quadrupole-quadrupole-time-of-ight; CE: Capillary Electrophoresis; OC: oral cancer; PC: pancreatic cancer.

(53) (Table 1) through a microarray platform. Amidst

their results, it was found that 1,402 genes had > 2 fold
up-regulation, while 2,447 > 2 fold down-regulation
in their saliva samples. Their findings indicate that
saliva is a relatively good source of transcripts for

biomarker discovery when comparing affected subjects

to healthy ones. A further selection of the 27 top upregulated candidates based on p-value and fold-change
(p < 0.01, fold change > 10) allowed them to select
potential candidates, that in the next stage of their

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study were validated through RT-PCR. This last assay

rendered 11 positive genes out of 27 genes. To be able
to complete their study, the 11 candidates were tested
in an independent sample (30 BC patients, 63 controls),
having found 8 pre-validated markers (53).
Microarray is a platform that despite its limitations
or a lower resolution compared with RNAseq, the
new high throughput platform for transcriptomics,
can be still very useful for studies in saliva, as it has
been demonstrated in the reviewed papers using this
approach in miRNA ESCC profiles. If a search over
the studies that focused on ESCC and microarrays
is performed directly in Gene Expression Omnibus
(GEO) using the keywords "ESCC" and "array", the
outcome gives back more than 300 results. However,
up to date and to our knowledge, no study in the
salivary ESCC transcriptome has been performed using
this platform yet, according to the last bibliographic
searches performed using keywords as "saliva",
"microarray", and "ESCC" or "oesophageal squamous
cell carcinoma". The exception to that comes from
several studies that focused in miRNA characterization
and identification using salivary samples (as well as
tissues).
In one of those studies, Ishibashi and colleagues (65)
used a microarray platform to analyse ESCC expression
profiles between normal and cancer affected samples
from 12 individuals. The 24 paired samples come either
from the tumour area with more than 80% of tumour
cells, or from normal tissue at least 4 cm away from
the affected zone. An interesting option for microarray
analysis in ESCC, has been the comparison of tumour
cells with cell lines overexpressing an important gene
for tumour progression (66), or a different splice variant
(67), as it can aid to clear many questions about the
development of the disease. Both of these studies have
been carried out in the same laboratory, and show the
potential of transcriptomics, as well as other type of
complementary technologies, to address fundamental
questions in molecular biology being an alternative
for obtaining a higher resolution profile than classic
approaches.
Identification of miRNAs in ESCC has been a
promising research area with a number of studies
published in the field. Matushima et al. (68) have
studied ESCC cell lines that were moderate and well
differentiated, as well as a control squamous epithelial
oesophageal line, through a microarray platform for
miRNA. Besides, they have performed functional
studies increasing or decreasing the expression of miR205, a miRNA overexpressed exclusively in ESCC
cell lines. Additional assays included estimation of
wound healing, cellular invasion and migration, or
evaluation of the regulation target, that in this case was
zinc finger E-box binding homeobox 2 (ZEB2). An
important feature of the work of Guo and collaborators
(69), is that they obtained the distinct miRNA profiles

for ESCC tumour samples in frozen archival tissues,

having obtained 46 differentially expressed ones. They
established a minimum set of 7 that can differentiate
between cancer and normal tissues. Despite the
degradation, miRNA profiles in these stored samples
were displaying similar values to that of fresh tissues,
enlightening the use of the extensive tissue archives
to gain better understanding of the disease. Some
significant examples with saliva samples can be found
in the recent work of Xie and collaborators (70), where
they found different miRNA profiles in saliva derived
from healthy patients and Oesophageal Cancer (EC)
affected individuals, with four of them validated in
a set of independent individuals, through a miRNA
microarray. According to them, their work has been the
first to assess the miRNA content in EC. Comparing
both of the above mentioned studies, it seems that
saliva renders less information, though is still valuable
resource for biomarker discovery in terms of miRNAs.
As it was pointed out by Xie (70), saliva can be
considered an end point of blood circulation, having
a certain degree of representation of the molecules in
blood, stating that it serves for diagnostic purposes.
Further broadening this point, Wang and collaborators
(71) carried out a meta-analysis in several papers
studying ESCC miRNA profiles in Asian populations,
including that of Xie et al. (70). They observed that
miRNA profiles in blood have a bigger diagnostic
accuracy than those derived from saliva. Despite this
observation, miRNA based diagnostics is a promising
field in saliva (as well as blood), due to the higher
stability, reproducibility, correlated concentrations to
some types of cancers, or ease of detection through RTqPCR (71).
Within the new RNAseq methodologies there
have been promising discoveries focusing on saliva,
as the work of Spielmann and colleagues (43) (Table
1), in which they highlight the power of salivary
transcriptome as a diagnostic tool for human diseases
using a massive RNA sequencing approach. With a
similar methodological approach, Franzosa (72) (Table
1) revealed the importance of RNA studies in related
locations to ESCC within human body by studying
human oral and gut microbiome and transcriptome. The
work of Spielmann and collaborators (43) is the first
whole RNAseq in saliva samples from healthy human
subjects, broadening to new studies in the field that will
focus on the differences between healthy and affected
samples. Both types of approaches have not been used
in ESCC yet, and could therefore pose a great source of
different biomarker tools. As it was mentioned above, the
oral microbiome is an important part to consider when
studying saliva. Spielmann and collaborators (43) report
a 20-25% of sequenced reads that align to the human
genome, while another 30% aligns to the Human Oral
Microbiome Database (HOMD). They have detected
more than 4,000 genes (coding and non-coding), while

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structural integrity of the transcripts was conserved
(43). Considering the potential of the metabiome as
a diagnostic tool, there has been a close study in the
oral metabiome between healthy and affected patients
of periodontal disease (73) (Table 1), having found
differences in the diversity of the community in both
cases. A very interesting suggestion was that metabolic
pathways are conserved while there are geographical,
ethnical, and food consuming factors that may alter
the species presence. If this gets demonstrated, we
may expect that microbiome in ESCC affected patients
displays different expression patterns to that one
observed in healthy subjects, and this could serve as a
prognostic marker for the development of the disease.
Another study using RNAseq in salivary samples was
performed afterwards by Bahn and colleagues (74)
(Table 1). They carried out a similar massive study on
the cell free component of salivary samples than that
of Spielmann (43), but focused on extracellular RNAs
(exRNAs) as micro RNA, Piwi-interacting RNA, and
circular RNA.
3. Proteomics
The development of proteomics has been notable in
different body fluids as urine, blood and saliva, as
Amado and colleagues (75) have indicated. One of
the features that they highlighted was the convenience
of adding proteases inhibitors, due to the presence of
proteases from bacteria and saliva which may affect
the downstream procedure. As it has been already
mentioned in the introduction, most of the proteins in
saliva (90%) are shared with blood, but they represent
only 15% w/w (41), while there have been studies
reporting that 20-30% of the proteins in saliva can be
found also in plasma (45,46). An interesting question
that arises in here, and in agreement with Amado (75),
is about how many of those proteins belong to the
human moiety, or to the metabiome. An answer to that
question was obtained by Jagtap and collaborators (76)
(Table 1), in a deep study of the salivary metaproteome
of healthy subjects, where they found that most of the
detected proteins had a human origin, being non-human
peptides present in much lower quantities. Additionally
they determined over 200 different bacterial species.
Their focus was the salivary supernatant, which
largely remains free of bacterial component, as the
pellet fraction collects the bacteria present in the oral
cavity after the initial centrifugation. Even though, by
using pooled samples from 6 individuals, they were
able to find bacterial peptides in this fraction. Besides
these significant discoveries, their study can serve as
a reference for future studies that will focus on the
differences between healthy subjects and those affected
by a disease, especially ESCC. An extensive review
on the topic was written by Uemura and collaborators
(77), which focuses on the proteomics advances

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regarding EC in its two main forms, ESCC and EAC.

This thorough revision of the bibliography, gives a
precise idea of the status until 2014 of the usage of
proteomics for different downstream applications, such
as early detection, prediction of lymph node metastasis,
therapy response prediction, prognostic prediction,
as well as the applications in the development of
novel therapeutics, or the elucidation of molecular
mechanisms of action. Although all of those studies
reviewed by Uemura are focused mainly on serum or
biopsy samples from ESCC or EAC tissues, they give a
complete perspective on what has been done until date
using the available proteomic approaches. Equally as
interesting, is the review written in 2012 by Qi et al. (78)
which focused exclusively on ESCC proteomics, yet
there were no reported studies using saliva as the source
for proteins.
One of the included studies in the paper from
Qi, was a proteomic profiling of cancer tissues from
Chinese ESCC subjects, carried out by Du and
collaborators (79), who found differential expression
of 22 proteins (17 up- and 5 down-regulated) through
MALDI-TOF (Matrix-Assisted Laser Desorption/
ionization- Time of Flight) or LC-ESIT-IT MS (Liquid
Chromatography-Electrospray/Ionization Ion Trap)
approaches. According to Uemura (77), this work can
be classified into the group of prognostic prediction
biomarkers, considering that one of their findings was a
correlation between poor prognosis and the expression
levels of calreticunin, and 78-kDA glucose-regulated
protein (GRP78) (79). The biological functions that
were represented in the differentially expressed proteins
are related with terms as glycolysis, regulation of
transcription, cell proliferation, cell motility or cell
signal transduction among others, what is related with
the kind of processes that occur within a group of
malignant cells, for example the Warburg effect (80).
But coming back to the field of salivaomics,
one interesting approach was followed by Hu and
colleagues, who used saliva as the source material to
study the proteome of 64 healthy subjects compared
with another group of 64 affected patients, although
the disease in this case was OSCC (81) (Table 1).
The methodology included a subtractive proteomics
approach for their in-depth study, through a combination
of separation techniques such as LC and LC-MS/MS,
together with a QqTOF MS. About the methodologies,
further explanation of the techniques can be consulted
in the two above mentioned reviews from Uemura (77)
and Qi (78). In their study Hu and colleagues concluded
with a verification of their experimental results, being
promising targets for biomarker discovery in OSCC.
Regarding saliva and ESCC, we were not able to find
studies relating both, only the reported ones using saliva
in related affections as OSCC. This situation makes
proteomic biomarker discovery in ESCC a promising
and unexplored field of research.

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4. Metabolomics
A remarkable review about the application of
metabolomics for biomarker discovery can be found
in the work of Armitage and Barbas (82). They have
highlighted a variety of key pathways that are altered
in cases of cancer, but considering all the potentially
involved metabolites there is no current platform
that can detect all of them at the same time. The
choice of analytical platform can range between the
two main techniques used in metabolomics analysis,
one of them being MS (Mass Spectrometry) based
approaches, with a deep profiling capacity; and NMR
(Nuclear Magnetic Resonance). The latter has the
advantage of being fast and reproducible (83), while
there is no need to disrupt the tissues or samples, a
feature that can be used for in vivo studies as showed
by Morvan and Demidem (84), where they analysed
the response of tumours to a chemotherapy treatment
in tissues and mice. Albeit these advantages, it shows
lower resolution than MS based choices, for which
there have been advances in separation techniques
with the coupling of MS to different complementary
methodologies, for example CE-MS (Capillary
Electrophoresis), GC-MS (Gas Chromatography), or
LC-MS (Liquid Chromatography) (82).
Unfortunately, up to date there have been no
published studies on metabolite analysis in saliva
of ESCC patients. However some works as that of
Sugimoto et al. (85) (Table 1), used the saliva of
subjects affected in breast (30 subjects), oral (69
subjects), and pancreatic cancers (18 subjects), as well
as periodontal disease (11 subjects), compared with a
set of 87 healthy ones. In their approach they used a
combination of CE-TOF-MS methodologies, having
found a set of 57 metabolites for accurate prediction
of the probability of each disease. Moreover, they
show that it is possible to obtain valuable metabolomic
information and biomarkers from saliva, in a variety
of cancer diseases that affect other areas of the human
body than oral cavity. One of the important metabolites
discovered, choline, is relevant because cholinecontaining metabolites participate in phospholipid
metabolism of cell membranes, and that has been
associated to malignancy, as it has been reported
by other authors that is a reflection of the increased
proliferation state of tumorigenic cells.
A recent review has been published by Abbassi
Ghadi (86) focused on studies using any type of
sample in gastric and oesophageal cancers, being the
most interesting studies for ESCC salivaomics, those
performed in biofluids as serum or urine. The variety
of analytical platforms from the studies included in
this review range from GC-MS, High ResolutionMagic Angle Spinning-NMR, CE-MS, LC-MS, and
Selected Ion Flow Tube-Mass Spectrometry. Due to
the inherent differences in each platform, sensitivity,

sample preparation, or type of sample source, there was

a notable variability among the reviewed references,
having found that glutamine is the most consistent
biomarker for both cancers across many studies. An
interesting idea highlighted by these authors is that
potentially useful biomarkers should be further tested in
other analytical platforms, and using different statistical
approaches, to lower as much as possible the false
positives discovery.
Wu and colleagues (87) focused on the screening of
20 paired samples from the same patients affected by
EC (18 ESCC and 2 EAC), including both non-affected
tissue and tumour samples (with at least 90% cancer
cells), resulting in the identification of 20 metabolites
through GC-MS. Other metabolomics studies focusing
exclusively on ESCC as the disease model, include
the one from Yang and collaborators (88), where they
have studied the profile changes in ESCC tumours at
different stages derived from tissue samples using a
NMR based approach. They have addressed, according
to their results, the possibility that some metabolic
changes arises before any morphological alterations
could be detected, and that is precisely what could pose
an advantage in the early screening of this disease. Jin
and collaborators (89) have pointed out that ESCC
metastasis advances primarily through the lymphatic
system, acting as well as a key prognostic factor. In
their study they used GC-MS to elucidate the possible
alterations in a set of ESCC serum samples (including
metastatic and non-metastatic ones), versus healthy
controls. One of the key elements of their study was
the evaluation of the metabolomics differences in those
subjects with lymph node metastasis. They have found
a marked Warburg effect (80) on ESCC cells due to the
enhanced glycolysis, which leads to decreased levels
of glucose and glutamine in blood, as well as a notably
higher content of lactic acid. This last metabolite was
found in higher quantities in those patients with lymph
node metastasis than non-metastatic ESCC subjects.
Some other metabolites usually associated with tumour
cells, are for example the observed increased levels
of certain long chain fatty acids, that could be derived
from a stronger de novo fatty acid biosynthesis, with
some fatty acids being up-regulated in the metastatic
samples compared with non-metastatic ones. Glutamine
is another metabolite that was found to be decreased
in metastatic cells, at its lowest levels from the three
groups. They demonstrated that a combination of
altered metabolites in cancer cases, could be used as
a metabolomic signature for discrimination between
patients in different stages.
Xu and collaborators (90) studied the metabolomics
differences in ESCC, through a RR-LC-MS (Rapid
Resolution) platform. They analysed different blood
samples from healthy and affected patients, as well
as other samples derived from ESCC patients that
underwent a chemoradiotherapy treatment, being

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divided in two groups, Overall Responders and
non-Overall Responders. Among their results, 11
of the discovered metabolites were classified as
tentatively potential biomarkers, while another set
of 18 metabolites were classified in other group that
potentially will serve as biomarkers for the diagnosis of
ESCC. In keeping with the results from the study of Jin,
there was an observation of an abnormality in the levels
of several fatty acids. A similar finding was reported by
Liu et al. (91), who used peripheral blood in order to
isolate cell-free plasma through a UPLC-ESI-TOF-MS
(ultraperformance liquid chromatography-electrospray
ionization-accurate time-of-flight) platform, having
found 6 metabolites related with the phospholipids
metabolism, out of 11 potential metabolite biomarkers.

Acknowledgements
This work has been funded by the Ministry of
Education and Science of the Republic of Kazakhstan.
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5. Conclusion
Despite the potential drawbacks that saliva may have,
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(Received July 3, 2015; Revised August 1, 2015; Accepted
August 25, 2015)

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