Expert Review of Molecular Diagnostics

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Serum, plasma and saliva biomarkers for head and

neck cancer

Lidia Maria Rebolho Batista Arantes, Ana Carolina De Carvalho, Matias

Eliseo Melendez & André Lopes Carvalho

To cite this article: Lidia Maria Rebolho Batista Arantes, Ana Carolina De Carvalho, Matias
Eliseo Melendez & André Lopes Carvalho (2018) Serum, plasma and saliva biomarkers
for head and neck cancer, Expert Review of Molecular Diagnostics, 18:1, 85-112, DOI:
10.1080/14737159.2017.1404906

To link to this article: https://doi.org/10.1080/14737159.2017.1404906

Accepted author version posted online: 14

Nov 2017.
Published online: 20 Nov 2017.

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EXPERT REVIEW OF MOLECULAR DIAGNOSTICS, 2018
VOL. 18, NO. 1, 85–112
https://doi.org/10.1080/14737159.2017.1404906

REVIEW

Serum, plasma and saliva biomarkers for head and neck cancer
Lidia Maria Rebolho Batista Arantes, Ana Carolina De Carvalho, Matias Eliseo Melendez and André Lopes Carvalho
Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos – SP, Brazil

ABSTRACT ARTICLE HISTORY

Introduction: Head and neck squamous cell carcinoma (HNSCC) encompasses tumors arising from Received 14 July 2017
several locations (oral and nasal cavities, paranasal sinuses, salivary glands, pharynx, and larynx) and Accepted 10 November 2017
currently stands as the sixth most common cancer worldwide. The most important risk factors identified KEYWORDS
so far are tobacco and alcohol consumption, and, for a subgroup of HNSCCs, infection with high-risk DNA methylation; head
types of human papillomavirus (HPV). Despite several improvements in the treatment of these tumors and neck cancer; lncRNA;
in the last decades, overall survival rates have only improved marginally, mainly due to the advanced microRNA; mRNA; plasma;
clinical stage at diagnosis and the high rates of treatment failure associated with this late diagnosis. protein expression; saliva;
Areas covered: This review will focus on the feasibility of evaluating molecular-based biomarkers serum
(mRNA, microRNA, lncRNA, DNA methylation and protein expression) in body fluids (serum, plasma,
and saliva) as markers for diagnosis, prognosis, and surveillance.
Expert commentary: The potential use of those markers in the clinical setting would allow for early
diagnosis, prediction of treatment response, improvement in treatment selection and provide disease
monitoring for early detection of tumor recurrence. It can ultimately be translated into better survival
rates and improved quality of life for HNSCC patients.

1. Introduction undiagnosed until they have reached an advanced stage when

damage is irreversible and treatment is inefficient.
Head and neck squamous cell carcinoma (HNSCC) encompasses
Liquid biopsy is a biofluid test of materials such as plasma,
tumors arising from the nasal cavity, paranasal sinuses, oral
serum, and saliva that detects circulating tumor cells (CTCs) or
cavity, salivary glands, pharynx, and larynx [1]. HNSCC currently
circulating cell-free tumor DNA (cfDNA) which are shed into the
stands as the sixth most common cancer worldwide with
bloodstream by cancer cells undergoing apoptosis or necrosis
900,000 new cases and 300,000 deaths annually [2]. The most
[7]. Compared to tissue biopsies, body fluids remain the best
important risk factors are tobacco and alcohol consumption,
choice for disease screening, diagnosis, and monitoring
which seem to have a synergistic effect. A subgroup of HNSCCs,
because of the advantages in accessibility, low invasive proce-
particularly those of the oropharynx, is caused by infection with
dure, low cost, and possibility of multiple sampling for monitor-
high-risk types of human papillomavirus (HPV) [3], while
ing the disease development [8]. Up to date, there have been
Epstein–Barr virus infection is associated with the development
hundreds of studies about HNSCC biomarkers identified in body
of nasopharyngeal tumors [4]. While the prevalence of smoking
fluids. The most popular body fluids which may contain reliable
and alcohol consumption has continued to decline over the
biomarkers for detecting HNSCC include peripheral blood and
past decade, the global incidence of HNSCC is on the rise, and
saliva [9]. This review will elaborate on the potential utility of
this has been attributed to an increase in HPV infection [5].
evaluating promising molecular-based biomarkers in body
Improved diagnostic accuracy for HNSCC could lead to earlier
fluids (serum, plasma, and saliva) as a tool to improve the
diagnosis, increasing patient survival rates.
diagnosis, prognosis, and surveillance for HNSCC patients.
The search for disease-specific biological features (namely
biomarkers) in patient samples such as tissues and body fluids
through molecular biology techniques is known as molecular 2. Plasma and serum as sources for liquid biopsy
diagnostics. The use of biomarkers in a clinical setting may aid
Plasma is the liquid portion of the blood and serves as a
in early diagnosis, predict tumor response, guide therapy selec-
transport medium for delivering nutrients to the cells of the
tion, and aid in patient’s follow-up, leading to increased survival
various organs of the body and for transporting waste pro-
rates and improved quality of life. Currently, tissue-based diag-
ducts for excretion. It is also a transport system for blood cells
nostic strategies require the test of materials obtained by inva-
and plays a critical role in maintaining normal blood pressure
sive procedures such as biopsies or needle aspirations commonly
[10]. If all the cellular elements from whole blood are removed
associated with substantial patient discomfort and health-care
by centrifugation, the resulting liquid is the plasma which is
costs [6]. Moreover, it is well known that molecular alterations
mainly constituted of water (92%). The dissolved solids in the
precede clinical symptoms; therefore, many disorders remain

CONTACT André Lopes Carvalho carvalhoal@gmail.com Molecular Oncology Research Center, Barretos Cancer Hospital. Rua Antenor Duarte Villela, 1331,
Bairro Dr. Paulo, Zip code: 14784-400, Barretos – SP, Brazil
© 2017 Informa UK Limited, trading as Taylor & Francis Group
86 L. M. R. B. ARANTES ET AL.
plasma are mainly proteins (7%) and the remaining 1% con-
tains glucose, lipids, enzymes, vitamins, hormones, and waste
products such as urea and CO2 [11].
Serum is a clear liquid that can be separated from clotted
blood. Its composition is the same as plasma, except for the
fibrinogens, a protein that is involved in blood coagulation.
Serum includes all proteins not used in blood clotting and all
the electrolytes, antibodies, antigens, hormones, and any exo-
genous substances. Blood is centrifuged to remove cellular
components. Anti-coagulated blood yields plasma containing
fibrinogen and clotting factors. Coagulated blood (clotted
blood) yields serum without fibrinogen [10].
Increased level of circulating nucleic acids (DNA, messenger
RNA [mRNA], and microRNA [miRNA]) has been observed in the
blood of patients with pathologic processes. Additional genetic
and epigenetic changes have been detected on circulating DNA
which play major part in tumorigenesis and progression.
Detecting cell-free nucleic acids (cfNA) in plasma or serum could
serve as a ‘liquid biopsy,’ which would be useful for numerous
diagnostic and monitoring applications. The use of such liquid
biopsy delivers the possibility of taking repeated blood samples,
consequently allowing the changes in cfNA to be traced during
the natural course of the disease or during cancer treatment [12].
The physiological events that lead to the increase of cfNA
during cancer development and progression are still not well
understood. However, analyses of circulating DNA allow the
detection of tumor-related genetic and epigenetic alterations
that are relevant to cancer development and progression. The
release of nucleic acids into the blood is thought to be related
to the apoptosis and necrosis of cancer cells in the tumor
microenvironment [12]. Tumors usually represent a mixture of
different cancer cell clones (which account for the genomic and
epigenomic heterogeneity of tumors) and other normal cell
types, such as hematopoietic and stromal cells. Thus, during
tumor progression and turnover, both tumor-derived and wild-
type (normal) cfNA can be released into the blood [12].
3. Saliva as a source for liquid biopsy
Saliva is an acidic biological fluid composed of secretions from
the salivary glands, gingival crevicular fluid, cell debris, plaque,
bacteria, nasal and bronchial secretions, lining cells, blood,
and exogenous substances [13–15]. Saliva plays an important
role in many biological functions such as perception of oral
sensations, lubrication, chewing, swallowing, and digestion
and protects oral mucosa against biological, mechanical, and
chemical factors, as well as against bacterial, viral, and fungal
infections [16,17]. It can be easily collected in a fast, inexpen-
sive, and noninvasive way [18] and can reflect the current
physiological state of an individual [13,19]. Interestingly, sali-
vary biomarkers signal not only for oral disorders but also for
pathologies in distal tissues and organs, suggesting that oral
fluids may represent a substantial reservoir of molecular and
microbial information capable of communicating the onset or
presence of disease throughout the body [6]. Tumors within
the oral cavity may shed cellular material directly into saliva
which allows it to be considered as a ‘sampling matrix’ for
many molecular and physiological processes and may reflect
aberrant pathological changes that occur in distant organ
systems [20]. Given the potential of this body fluid for use in
a clinical setting, a growing number of studies are focused on
elucidating and developing saliva-based biomarkers indicative
of both local and systemic diseases [6].
4. Detection of RNA-based biomarkers in body fluids
for the management of HNSCC
The transcriptome is composed of all coding (mRNA) and
noncoding (such as small nucleolar RNAs, small nuclear
RNAs, long-noncoding RNAs, and miRNAs) transcripts present
in a cell at a specific developmental stage or physiological
condition. It helps to reveal the functional elements of the
genome as well as molecular components of cells and tissues,
development, and disease [21], being a significant source of
potentially relevant biomarkers. The main method for identifi-
cation of transcriptomic biomarkers is microarray that can be
validated by means of the quantitative real-time polymerase
chain reaction PCR (qPCR). More recently, RNA-sequencing
platforms have been used for the high-throughput assessment
and quantitation of differentially represented transcripts,
allowing for a more thorough understanding of the transcrip-
tome in different samples, including body fluids.
A major challenge in the use of RNA quantification levels as
biomarkers in the clinics is the lack of consistent reports
regarding its usefulness for this purpose. The lack of reprodu-
cibility might be due to a lack of multicenter studies and
sufficiently powered cohorts. Moreover, there is also a wide
variation in the techniques used such as differences in sample
types and sources (saliva, mouth wash, plasma, serum, and
whole blood); the moment these samples were obtained (pre-
or posttreatment and follow-up visit); purification (either from
exosomes or from whole serum/plasma/saliva) and extraction
methods (Trizol and commercial kits); processing time and
protocol and storage of the fluids and nucleic RNA; differences
in the methods used for quantification; and the choice for data
normalization since there is no consensus about a suitable
endogenous reference to be used in biological fluids. The
blood collection and processing is a particularly sensitive
step: RNA contamination can occur during sample collection;
the time elapsed between blood collection and processing
should be minimized to prevent lysis and cellular contamina-
tion; the type of anticoagulant used in plasma collection can
influence downstream technologies; and so on. Another
important issue is the difficulty in accurately quantifying RNA
in samples from biological fluids due to the low quantities of
these molecules present in these samples and the high levels
of contaminating salts and proteins that can interfere with
spectrophotometric measurement. For this reason, studies
often use fixed volumes of starting material to standardize,
even if it is evident that they may contain different amounts of
RNA. These issues highlight the need to standardize experi-
mental conditions for circulating RNAs studies, as well as the
need to validate these findings in additional independent
cohorts as well as preclinical/clinical verification studies,
before the clinical utility of circulating RNAs may be estab-
lished to avoid biased results, mainly when using this
approach for disease monitoring and treatment decisions (as
reviewed in [22]).

4.1. Messenger RNAs
mRNAs take the message encoded in the DNA within genes to
the cytoplasm to be translated into proteins. mRNAs testing as
diagnostic and prognostic markers in body fluids of HNSCC
patients have been little explored, given the known low sta-
bility of these markers due to their high susceptibility to RNase
degradation in body fluids, hindering their efficacy and repro-
ducibility as liquid-biopsy markers (Table 1). However, Li and
colleagues shed light into this matter and showed the feasi-
bility of assessing mRNA in saliva samples. They evaluated the
transcriptome of saliva samples from 32 primary oral squa-
mous cell carcinoma (OSCC) samples and 32 normal matched
individuals and found that a large panel of human mRNAs can
be reliably detected in saliva. Seven cancer-related mRNA
biomarkers were found upregulated in the saliva from cancer
patients and were consistently validated. These potential sali-
vary RNA biomarkers are transcripts of IL8, IL1B, DUSP1, HA3,
OAZ1, S100P, and SAT. Four salivary mRNAs (OAZ, SAT, IL8, and
IL1b) collectively showed a discriminatory power of 91% sen-
sitivity and specificity for oral cancer detection, demonstrating
the utility of salivary transcriptome diagnostics for oral cancer
detection [23]. Given these promising findings, other studies
have tried to validate these findings in other cohorts and
applications. A study evaluated whether the seven mRNAs
described earlier (IL8, IL1B, DUSP1, HA3, OAZ1, S100P, and
SAT) were capable of discriminating patients with OSCC from
healthy subjects in five independent case-control cohorts
including 395 subjects. Expression of all seven mRNAs was
increased in OSCC versus controls in all five cohorts and the
increase in IL-8 and SAT was statistically significant and
remained top performers across different cohorts in terms of
sensitivity and specificity, thus validating previous findings
from Li and colleagues [24]. More recently, a study checked
for the presence of OAZ, SAT, IL8, and IL1b in the saliva of 34
patients with primary OSCC (T1N0M0/T2N0M0), 20 patients
with oral leukoplakia and dysplasia, and 31 matched healthy
control subjects as a tool for the early detection of OSCC. The
results confirmed a good predictive probability of 80% for
patients with OSCC but not for patients suffering from oral
potentially malignant disorders. Moreover, the combination of
only the two biomarkers (SAT and IL-8) also showed a high
predictive ability of 75.5%, suggesting the use of the two
biomarkers only in the prediction model for OSCC patients,
limiting the economic and health costs [25].
Other relevant studies confirmed the feasibility of assessing
mRNA in body fluids; however, they are not numerous [26–28].
Aggarwal and colleagues found that OSCC patients expressed
significantly higher levels of gal-1 and gal-3 mRNA in serum
and at the tumor sites than controls. Moreover, patients with
higher tumor load showed significantly higher expression of
both galectins which were able to discriminate between
healthy subjects and patients with more than 80% sensitivity
and specificity [26]. Another study evaluated the levels of
transgelin mRNA in tissue, serum, and saliva of OSCC patients
and negative controls. Little difference of serum transgelin
mRNA expression was found between the OSCC patients and
healthy controls; however, this marker was elevated in tissue
and saliva from OSCC patients. These high levels were
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 87
correlated with clinicopathological parameters such as poorer
overall survival and were independent prognosis factors for
OSCC, indicating that they might serve as promising biomar-
kers for OSCC [27].
HPV has been recently described as an important risk factor
for HNSCC tumors, especially those in the oropharynx. The
presence of HPV-16 RNA in salivary oral rinses of 82 HNSCC
patients with tumor p16(INK4a) status was evaluated: of 40
patients with p16(INK4a)-positive tumors, HPV-16 mRNA was
detected in 24 and no HPV-16 mRNA was detected in oral
rinse samples from the p16(INK4a)-negative patients [28].
Noncoding RNAs such as miRNAs and long noncoding
RNAs, although transcribed from genes, do not code for pro-
teins and act as regulators of the expression of coding genes.
These molecules are promising candidates for biomarkers
since they circulate stably in human body fluids and can be
obtained with noninvasive methods [29].
4.2. MicroRNAs
miRNAs are small regulatory (19–21 nucleotides) nucleic acids
that interact with and potentially silence the translation of
complementary antisense RNA sequences [30,31]. They play
an important role in cell growth, differentiation, apoptosis,
stress, and immune response [32]. Similar to mRNAs, miRNAs
can be aberrantly expressed and contribute to tumorigenesis
[33] by either enhancing the expression of oncogenes or
inhibiting the expression of tumor suppressor genes [34].
miRNAs may be universally present in several body fluids
and excretions [35]. These molecules are released passively
into body fluids as cell-free miRNAs, are associated with
RNA-binding proteins, or can be loaded into microvesicles or
incorporated in exosomes and then released in the extracel-
lular space [22,36–39]. Exosomes are microvesicles of
30–120 nm, which play an important role in intercellular com-
munication [40,41] and represent around 3% of the entire
amount of cell-free miRNAs [36]. Due to the exosome carriage
and binding to RNA-binding proteins, miRNAs have been
reported to be extremely stable in serum and plasma samples
[42]. Because of their tumor-specific expression, stability in
tissues, in the circulation, and in body fluids, and the possibi-
lity of being tested in small quantities rapidly and accurately,
miRNAs have been extensively tested and results suggest that
they might be used as cancer biomarkers in diagnostics, clas-
sification, and prognostic stratification of HNSCC [43–45].
4.2.1. Diagnosis
Several studies have assessed the potential of miRNAs as
diagnostic markers, suggesting a potential clinical application
for HNSCC-specific miRNA signatures in body fluids (Table 2).
Park et al. screened a total of 314 miRNAs in saliva from 50
OSCC patients and 50 healthy matched controls and found a
downregulation of miR-125a and miR-200a in patients with
oral cancer [46]. Another study describes an oral cancer-spe-
cific panel containing aberrant miR-375 and miR-200a expres-
sion and miR-200c-141 methylation which was able to
distinguish oral rinse and saliva from OSCC patient and
healthy volunteers [47]. Momen-Heravi and colleagues tested
 88
L. M. R. B. ARANTES ET AL.

Table 1. Summary of data from relevant mRNA-based markers in body fluids of head and neck tumors.
Biomarker Tumor Sample Technological
name Biomarker behavior site type Sample size approach Application Comments References
IL8, IL1B, Seven cancer-related mRNA biomarkers were OSCC Saliva Preliminary study: 32 Microarray, qPCR, and Diagnosis IL-8 and SAT were top performers across [23,24]
DUSP1, found upregulated in the saliva from cancer primary OSCC patients ELISA different cohorts in terms of sensitivity and
HA3, OAZ1, patients and were consistently validated. and 32 healthy specificity in discriminating cases from
S100P, and subjects controls.
SAT Validation study: 395
subjects from five
independent case-
control cohorts
OAZ, SAT, IL8, These markers showed a good predictive OSCC Saliva 34 patients with primary qPCR Diagnosis IL-8 and SAT alone showed a high predictive [25]
and IL1b probability for OSCC patients but not for and initial OSCC ability suggesting the use of the two
patients suffering from oral potentially 20 patients with oral biomarkers only in the prediction model for
malignant disorders. leukoplakia and OSCC.
dysplasia
31 matched healthy
subjects
Gal-1 and Gal- OSCC patients expressed significantly higher OSCC Serum 60 OSCC patients qPCR and Western blot Diagnosis A three-time higher risk of OSCC in subjects [26]
3 levels of gal-1 and gal-3 mRNA in serum and 30 healthy subjects over expressing these proteins was
tumor tissue. observed.
Transgelin This marker was elevated in tissue and saliva OSCC Serum 78 primary OSCC qPCR Diagnosis and Overexpression of tissue or salivary transgelin [27]
from OSCC patients, but not in serum. and patients and 78 prognosis was associated with clinical parameters
saliva healthy subjects including poorer overall survival.
p16(INK4a) Detection of HPV-16 mRNA in oral rinses from HNSCC Oral 82 HNSCC patients qPCR Diagnosis of HPV- The detection of HPV-16 mRNA was [28]
HNSCC with 100% specificity and 60% rinse positive tumors compared with p16(INK4a) status.
sensitivity.
HNSCC: head and neck squamous cell carcinoma; OSCC: oral squamous cell carcinoma; qPCR: quantitative polymerase chain reaction; HPV: human papillomavirus.
Only relevant findings were included in the table.
Table 2. Summary of data from relevant noncoding RNA-based markers in body fluids of head and neck tumors.
Sample Technological
Biomarker name Biomarker behavior Tumor site type Sample size approach Application Comments References
miR-125a and miR- Downregulation in OSCC OSCC Saliva 50 OSCC patients and qPCR Diagnosis [46]
200a 50 healthy subjects
(HS)
miR-375 and miR- Upregulation in OSCC OSCC Oral rinse 15 OSCC patients and qPCR Diagnosis [47]
200a and 7 HS
saliva
(i) miRNA-136, (i) Downregulation in OSCC; OSCC Saliva Nine OSCC patients NanoString nCounter Diagnosis MiR-136 was [48]
miRNA-147, (ii) upregulation in OSCC before treatment downregulated in
miRNA-1250, Eight patients with OSCC in comparison
miRNA-148a, OSCC in remission to OSCC and OSCC-
miRNA-632, (OSCC-R) R and mir-27b levels
miRNA-646, Eight patients with were higher in
miRNA-668, OLP OSCC than in OSCC-
miRNA-877, Nine HS R and OLP.
miRNA-503,
miRNA-220a, and
miRNA-323-5p; (ii)
miRNA-24 and
miRNA-27b
miR-9, miR-191, and Upregulation in OSCC HNSCC Saliva 56 HNSCC patients and miRNA microarray Diagnosis Salivary miRNA data [49]
miR-134 56 HS data, qPCR, in silico presented in the
validation (TCGA study showed a
data) good correlation
with the TCGA
miRNA data.
(i) miR-24, (ii) miR- Upregulation in OSCC OSCC Plasma (i) 33 OSCC patients qPCR Diagnosis [50–54]
181, (iii) miR-196a, and 10 HS; (ii) 39
(iv) miR-10b, and OSCC patients and
(v) miR-187* 12 HS; (iii) 65 OSCC
patients and 24 HS;
(iv) 56 OSCC
patients, 36 HS, and
7 patients with oral
precancer
leukoplakia; and (v)
63 OSCC patients
and
21 HS
miR-155 Upregulation in OSCC LSCC Plasma 280 LSCC patients and qPCR Diagnosis [55]
560 HS
miR-497 Downregulation in OSCC NPC Plasma 18 NPC patients and miRNA microarray Diagnosis [56]
11 HS (with chronic data, qPCR
nasopharyngitis)
miR-17, miR-20a, Upregulation in OSCC NPC Serum (i) 20 NPC patients and (i) miRNA microarray Diagnosis [57]
miR-29c, and miR- 20 HS data, (ii) qPCR
223 (ii) 30 NPC patients
and
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS

30 HS
(Continued )
89
 90

Table 2. (Continued).
Sample Technological
Biomarker name Biomarker behavior Tumor site type Sample size approach Application Comments References
miR-196a and miR- Upregulation in OSCC and precancer OSCC and Plasma 90 OSCC patients, qPCR Disease progression The combined [58]
196b patients premalignant 53 HS, and determination of
oral lesions 16 precancer patients miR-196a and miR-
196b levels
produces high
sensitivity and
specificity in the
diagnosis of
L. M. R. B. ARANTES ET AL.

patients with oral

precancer or oral
cancer, as well as in
the prediction of
potential
malignancy.
miR-21 Upregulation in PLL and LSCC LSCC and Plasma 76 LSCC patients, ddPCR Disease progression The study highlights [59]
premalignant 40 PLL patients, and the potential of
laryngeal lesions 19 HS ddPCR for clinical
use.
(i) miR-130b, (ii) miR- (i) Upregulation in nonmetastatic OSCC, OSCC Plasma 30 OSCC qPCR Prognosis [60]
296 (ii) upregulation in metastatic OSCC
miR-296 Upregulation in OSCC Metastatic OSCC Plasma qPCR Prognosis [60]
miR-142-3p, miR-186- Upregulation in HNSCC HNSCC Plasma 18 HNSCC and qPCR Prognosis The study found and [61]
5p, miR-195-5p, 12 HS validated circulating
miR-374b-5p, and promising markers
miR-574-3p for prognosis and
therapy monitoring
in the plasma of
HNSCC.
(i) miR-16, -21, -24, (i) Upregulation in NPC; (ii) NPC Plasma 217 NPC patients and qPCR Prognosis There was a negative [62]
and -155; (ii) miR- downregulation in NPC 73 HS (with chronic correlation between
378 nasopharyngitis) plasma miRNA
expression and
cancer progression,
where miR-21 was
statistically
significant in T and
N staging and miR-
16 and 24 were
significant in N
staging only.
miR-22, miR-572, Upregulation in NPC patients with a NPC Serum 512 nonmetastatic miRNA microarray Prognosis The study found that [63]
miR-638, and miR- poorer prognostic NPC patients data, qPCR the four-serum
1234 miRNA signature
may add prognostic
value to the TNM
staging system and
provide information
for personalized
therapy in NPC.
(Continued )
Table 2. (Continued).
Sample Technological
Biomarker name Biomarker behavior Tumor site type Sample size approach Application Comments References
miR-9 Downregulation in OSCC and OLK OSCC Serum 104 OSCC patients, qPCR Prognosis The study suggests [64]
30 OLK patients, and that miR-9 might
40 HS play a tumor-
suppressive role in
OSCC and can serve
as a promising
biomarker for this
disease.
miR-483-5p Upregulation in OSCC OSCC Serum 101 OSCC patients and miRNA microarray Prognosis miR-483-5p expression [65]
103 HS data, qPCR in serum is a
promising
diagnostic and
prognostic
biomarker for OSCC
miR-31 Upregulation in OSCC OSCC and oral Saliva; (i) 45 OSCC patients, qPCR Disease monitoring (i) miR-31 was more (i) [66]; (ii) [67]
premalignant plasma 10 oral verrucous abundant in saliva
lesions leukoplakia, and than in plasma.
24 HS
(ii) 43 OSCC and
21 HS
miR-139-5p Downregulation in TSCC TSCC Saliva 25 TSCC patients (pre- miRNA microarray Disease monitoring The authors suggest [68]
and posttreatment) data, qPCR that saliva can be
and used as a feasible
25 HS source for routine
TSCC diagnostics.
miR-184 Upregulation in TSCC TSCC Plasma 30 TSCC patients and qPCR Disease monitoring [69]
38 HS
miR-221 Upregulation in LSCC LSCC Plasma 30 LSCC patients qPCR Disease monitoring Plasma miR-221 may [70]
(preoperative and have a potential as
postoperative a novel diagnostic/
samples) and prognostic marker
30 HS in LSCC.
miR-21 and miR-26b Upregulation in HNSCC HNSCC Plasma 50 HNSCC patients and qPCR Prognosis; disease [71]
36 HS monitoring
(Continued )
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS
91
 92

Table 2. (Continued).
Sample Technological
Biomarker name Biomarker behavior Tumor site type Sample size approach Application Comments References
miR-BART7 and miR- Upregulation in NPC NPC Plasma 89 NPC patients (for 41 qPCR Prognosis; disease These results indicate [72]
BART13 patients, samples monitoring that EBV microRNAs,
were collected miR-BART7, and
L. M. R. B. ARANTES ET AL.

before and after miR-

radiotherapy) and BART13 may
28 HS constitute useful
new serological
biomarkers for
diagnosis of NPC
and prediction of
treatment efficacy
EBV microRNAs, miR-
BART7, and miR-
BART13 may
constitute useful
new serological
biomarkers for
diagnosis of NPC
and prediction of
treatment efficacy.
miR-9 Downregulation Advanced NPC Plasma 294 NPC patients and qPCR Prognosis; disease [73]
109 HS monitoring
(i) MiR-99a; (ii) miR- (i) Upregulation in HNSCC after surgery; HNSCC Plasma Nine HNSCC patients miRNA microarray Prognosis; disease These circulating [74]
21 and miR-223 (ii) downregulation in HNSCC after (preoperative and data, qPCR monitoring miRNAs may serve
surgery postoperative) and as biomarkers to
9 HS evaluate the efficacy
of therapy and the
prognosis of HNSCC.
miR-425-5p, miR-21- Downregulation in HNSCC after HNSCC Plasma 17 HNSCC patients miRNA microarray Prognosis; disease The abundance of [75]
5p, miR-106b-5p, treatment (pre- and data, qPCR monitoring miR-425-5p and
miR-590-5p, miR- posttreatment) miR-93-5p in the
574-3p, and miR- plasma changed
885-3p during
radiochemotherapy
and may represent
novel biomarkers
for therapy
monitoring.
LSCC: laryngeal squamous cell carcinoma; HNSCC: head and neck squamous cell carcinoma; OSCC: oral squamous cell carcinoma; NPC: nasopharyngeal squamous cell carcinoma; PLL: premalignant laryngeal lesion; OLP: oral
lichen planus; OLK: oral leukoplakia; TSCC: tongue squamous cell carcinoma; qPCR: quantitative polymerase chain reaction; ddPCR: droplet digital PCR; TCGA: The Cancer Genome Atlas; TNM: tumor nodal metastasis; TCGA:
The Cancer Genome Atlas; TNM: tumor nodal metastasis; EBV: Epstein–Barr virus.
Only relevant findings were included in the table.

more than 700 miRNAs in saliva samples from OSCC patients
using the NanoString nCounter technology and identified 13
miRNAs that were differentially expressed in OSCC when com-
pared to healthy controls: 11 miRNAs were downregulated
(miRNA-136, miRNA-147, miRNA-1250, miRNA-148a, miRNA-
632, miRNA-646, miRNA-668, miRNA-877, miRNA-503, miRNA-
220a, and miRNA-323-5p) and 2 miRNAs were upregulated
(miRNA-24 and miRNA-27b) [48]. Salazar and colleagues tested
five miRNAs in saliva from healthy controls and HNSCC
patients and found that miR-9, miR-191, and miR-134 expres-
sion can serve as novel noninvasive biomarkers for the diag-
nostic of HNSCC with a good discriminatory power. These
results were further validated in a cohort of HNSCC miRNA
expression data from The Cancer Genome Atlas database
showing the potential of this panel as HNSCC diagnostic
biomarker [49].
The evaluation of miRNAs in plasma samples from tumors and
controls showed an upregulation of several miRNAs such as miR-
24, miR-181, miR-196a, miR-10b, and miR-187* in OSCC tissues
and plasma relative to control samples [50–54] and an elevated
expression of miR-155 in tissue and plasma samples of laryngeal
squamous cell carcinomas (LSCC) than in those of the controls
[55]. A reduced expression of miR-497 was observed in the tissue
and plasma of nasopharyngeal squamous cell carcinoma (NPC)
patients relative to the plasma of noncancerous control patients
[56], while miR-17, miR-20a, miR-29c, and miR-223 were differ-
entially expressed in the serum of NPC when compared with that
of non-cancerous control [57].
4.2.2. Disease progression
The use of these molecules has also proved feasible as markers
for disease progression from oral premalignant lesions to
cancer (Table 2). The diagnostic efficacy of miR-196a and
miR-196b expression was assessed in plasma samples from
53 healthy individuals, 16 precancer patients, and 90 oral
cancer patients. Both circulating miR-196a and miR-196b
were substantially upregulated in patients with oral precancer
lesions and in oral cancer patients [58]. Besides, the expression
of miR-21 was found significantly higher in tissue and plasma
from LSCC and premalignant laryngeal lesions in comparison
with controls [59].
4.2.3. Prognosis
The evaluation of different miRNAs in plasma and serum from
HNSCC patients also showed associations with prognosis
(Table 2). MiR-130b was found upregulated in nonmetastatic
OSCC samples, and this was confirmed in plasma of patients
showing no metastasis, while miR-296 was detected in meta-
static tumors and the expression was confirmed in plasma of
patients presenting metastasis [60]. A high expression of a
panel of miRNAs namely miR-142-3p, miR-186-5p, miR-195-
5p, miR-374b-5p, and miR-574-3p in the plasma of HNSCC
patients correlated with worse prognosis [61]. Liu and collea-
gues found high levels of miR-16, -21, -24, and -155 in NPC
patients, whereas the level of miR-378 was decreased. There
was a negative correlation between plasma miRNA expression
and cancer progression, where miR-21 was statistically signifi-
cant in T and N staging and miR-16 and 24 were significant in
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 93
N staging only [62]. Another study performed a miRNA profil-
ing on the serum of NPC patients with shorter survival and
patients with longer survival matched by age, sex, and clinical
stage and the identified miRNAs were validated in 512 sam-
ples. Four serum miRNAs (miR-22, miR-572, miR-638, and miR-
1234) were differentially altered and were used to classify the
patients into high- or low-risk groups. Patients with high-risk
scores had poorer overall survival and distant metastasis-free
survival than those with low-risk scores [63].
A lower level of serum miR-9 was observed in patients with
OSCC and this was associated with T stage, lymph node
metastasis, and tumor nodal metastasis (TNM) stage.
Moreover, OSCC patients with low serum miR-9 expression
had poorer overall and disease-free survival rate, and this
marker remained an independent prognostic factor in multi-
variate analysis [64]. On the other hand, miR-483-5p expres-
sion was significantly increased in OSCC patients with a
significant correlation with TNM stage, lymph nodal metas-
tases, and lower survival than those with low expression.
Multivariate analyses for overall survival revealed that high
serum miR-483-5p expression was an independent prognostic
factor for OSCC, suggesting that this marker may be a novel
diagnostic and prognostic biomarker for OSCC [65].
4.2.4. Disease monitoring
One relevant use for body fluid-based markers is disease
monitoring.
In such studies, the levels of the markers of interest are eval-
uated before and after treatment and, when possible, during
patient follow-up (Table 2). Liu and colleagues analyzed the levels
of miR-31 in saliva of patients with oral carcinoma, premalignant
lesions, and healthy individuals and found this miRNA significantly
increased in patients with oral carcinoma at all clinical stages,
including very small tumors, but no change when comparing
the levels in premalignant lesions and healthy controls. After
excision of oral carcinoma, salivary miR-31 was remarkably
reduced, indicating that most of the upregulated salivary miR-31
came from tumor tissues [66]. This miRNA was also elevated in
plasma of OSCC patients and its level was remarkably reduced
after tumor resection, suggesting that this marker is tumor asso-
ciated [67]. The expression level of miR-139-5p was significantly
reduced in OSCC samples compared to the controls, and this level
was restored in the saliva after surgical removal of the primary
tumor [68]. Wong and colleagues evaluated the expression levels
of plasma miR-184 and found it significantly higher in tongue SCC
patients in comparison with normal individuals, and the levels
were significantly reduced after surgical removal of the primary
tumors [69]. In LSCC, miR-221 was found upregulated in samples
from cancer patients, and its level was found normal in post-
operative plasma samples [70]. The expression profiles of 10
miRNAs in plasma from 50 HNSCC patients and 36 healthy sub-
jects were evaluated. MiR-21 and miR-26b were significantly upre-
gulated in plasma samples obtained from patients, and levels of
both miR-21 and miR-26b were reduced after treatment in HNSCC
patients with good prognosis and remained high after tumor
removal in the expired cases [71].
Zhang and colleagues evaluated plasma specimens
obtained from NPC patients (n = 89), and healthy donors
94 L. M. R. B. ARANTES ET AL.

(n = 28), and non-NPC tumor patient controls (n = 18) and

References
found different levels of both miR-BART7 and miR-BART13,

[82]

[83]
[84]
with elevated levels being associated with advanced disease.
Moreover, the levels of this miRNAs decreased after radiother-

patients, while lincRNA-p21 and

apy [72]. Plasma microarray profiling identified 33 differentially

GAS5 levels were significantly

increased in nonresponder
expressed miRNAs between NPC patients and healthy volun-
teers. A low level of plasma miR-9 was significantly correlated

were not informative for

treatment response.
Comments
with worse lymphatic invasion and advanced TNM stage and
could distinguish locoregional from metastatic NPC cases with

HOTAIR levels
a high sensitivity and specificity. Furthermore, the plasma miR-

Response to treatment Pretreatment

9 level was significantly elevated in posttreatment plasma
compared with those pretreatment samples [73].
Another field of interest is to explore circulating miRNAs as

LSCC: laryngeal squamous cell carcinoma; HNSCC: head and neck squamous cell carcinoma; OSCC: oral squamous cell carcinoma; qPCR: quantitative polymerase chain reaction.
cancer therapy biomarkers (Table 2). Hou and colleagues com-

Diagnosis; prognosis
pared the miRNAs levels between pre- and 6 months post-

Application
operative paired plasma samples from nine HNSCC patients:
MiR-99a was found downregulated in cancerous tissues and

Diagnosis
significantly increased in plasma after operation; miR-21 and
miR-223 that were upregulated in cancerous tissues were
significantly reduced in postoperative plasma samples.

Semi-quantitative PCR
Furthermore, plasma miR-223 was inversely increased in a

Technological
patient whose cancer relapsed within 6 months after opera-

approach
tion [74]. In another study, miRNA profiles were analyzed in
paired plasma samples of HNSCC patients before therapy and
after two days of treatment. A panel of six miRNAs, namely

qPCR

qPCR
miR-425-5p, miR-21-5p, miR-106b-5p, miR-590-5p, miR-574-3p,
and miR-885-3p, significantly separated plasma samples col-

49 patients with polyps of vocal cords

41 HNSCC (pre- and posttreatment)

lected prior to treatment from plasma samples collected after
Table 3. Summary of data from relevant long noncoding RNA-based markers in body fluids of head and neck tumors.

two days of radiochemotherapy [75]. Worth mentioning, since

a decrease of tumor volume upon treatment will result in a
Sample size

decrease of total miRNA, the use of decreased levels of specific

miRNAs as predictive markers for treatment response should
39 healthy subjects
52 LSCC patients

be used carefully. In these cases, a possible way to reduce bias

is the use of the levels of miRNAs measured posttreatment as
a start point for comparisons of the amount of the markers in
9 OSCC

body fluids and monitoring of disease after the treatment.

Biomarker behavior Tumor site Sample type

4.3. Long noncoding RNAs (lncRNAs)

Plasma
Serum

Saliva

Long noncoding RNAs are also important regulators of gene

expression being involved in the regulation of several cellular
processes such as imprinting and maintenance of pluripotency
HNSCC
OSCC

and, as such, having an important role in tumorigenesis [76,77].

Upregulation in LSCC LSCC

Gene expression control is exerted either by direct regulation of

gene transcription, through the recruitment of gene silencing
complexes, precluding the access of regulatory proteins to DNA,
Only relevant findings were included in the table.
Upregulation
lincRNA-p21, GAS5, and HOTAIR Upregulation

modulating mRNA stability or as miRNA decoy [78–81]. Little is

known about the potential of these molecules as biomarkers and
they are an interesting field to be further explored in HNSCC.
Below, the few studies evaluating these markers in HNSCC with a
suggested clinical application are described (Table 3).
The levels of exosomal miR-21 and HOTAIR in serum of 52
LSCC patients and 49 patients with polyps of vocal cords were
evaluated. The expression of exosomal miR-21 and HOTAIR was
Biomarker name

significantly higher in patients with LSCC than those with vocal

cord polyps and associated with advanced T classifications and
MALAT-1

III/IV clinical stages. Moreover, patients with lymph node metas-

HOTAIR

tasis had higher serum exosomal miR-21 and HOTAIR expres-

sions than those without [82]. Tang and colleagues investigated

the relative abundance of a collection of lncRNAs in tissue and
saliva samples from OSCC patients and found subsets of
lncRNAs expressed across nontumor, tumor, and metastatic
tissue samples. Saliva samples seem to contain a detectable
amount of some lncRNAs, which appeared to be potential
markers for OSCC such as MALAT-1 which was present in all
the patients investigated, indicating that saliva may contain a
detectable amount of certain lncRNAs that may be potential
markers for OSCC diagnosis [83]. Moreover, plasma levels of
three lncRNA molecules (lincRNA-p21, GAS5, and HOTAIR) were
evaluated as markers for treatment response in HNSCC patients
treated with radical chemoradiotherapy. Pretreatment and
posttreatment GAS5 levels in patients with partial response/
progressive disease were significantly higher compared with
patients with complete response based on clinical investigation.
In contrast, pretreatment or posttreatment lincRNA-p21 and
HOTAIR levels were not informative for treatment response [84].
5. Detection of DNA-based biomarkers in body
fluids for the management of HNSCC
In the past decade, several studies have tried to depict the
genetic alterations involved in the initiation and progression of
head and neck tumors. The advantage of genetic alterations over
conventional biomarkers is that genetic changes are specific for
neoplastic cells. More recently, large-scale genomic studies have
shown that these tumors are characterized by high degree of
inter-tumor heterogeneity, confirming its complexity, and were
able to identify frequently mutated tumor suppressor genes and
oncogenes that could be involved in different signaling path-
ways critical for genomic integrity and epithelium differentiation
that may represent dominant genetic events in HNSCC carcino-
genesis [85]. Besides genetic alterations, the involvement of
high-risk HPV in HNSCC, mainly oropharyngeal tumors, has
started a parallel search for viral-induced alterations that could
be used as biomarkers, as well as the use of the detection of the
viral DNA itself as a biomarker [86].
Studies are now focusing on the utility of these cancer-
specific alterations as biomarkers for early diagnosis, therapy
response, and disease monitoring and surveillance [87]. For
this purpose, the first step is to characterize patient-specific
genetic mutations in the primary tumor. Next, the gene muta-
tion findings can be evaluated in body fluids collected from
the patient at different time points during follow-up after the
last curative treatment (chemo, radiotherapy, or primary
tumor resection) [88]. The detection of genetic mutations in
circulating tumor DNA (circulating tumor DNA [ctDNA]) or in
CTCs was proven feasible in body fluids and has shown a
clinical utility as prediction biomarkers in different cancer
types [89]. One challenge in exploiting these alterations in
body fluids is the low concentration of mutant alleles in
these samples; however, technological advances have made
it possible [86,87].
In HNSCC, a recent proof-of-concept study explored the
potential of tumor-specific DNA as a biomarker in plasma and
saliva of patients with tumors of various stages and anatomical
sites [86]. Tumor DNA released from the basal side of HNSCC
epithelial cells into the lymphatics or venous system should be
detectable in plasma, while DNA that is released primarily on
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 95
the apical side of HNSCC should be detectable in the saliva
[90,91]. DNA from saliva or plasma was tested for somatic
mutations on TP53, PIK3CA, CDKN2A, HRAS, NRAS, or HPV
genes (HPV-16 and HPV-18), and when both fluids were tested,
tumor DNA was detected in 96% of 47 patients (100% of early-
stage tumors and 95% of late-stage tumors). In saliva, tumor
DNA was found in 100% of patients with oral cavity cancers and
in 47–70% of patients with cancers of the other sites. In plasma,
tumor DNA was found in 80% of patients with oral cavity
cancers and in 86–100% of patients with cancers of the other
sites. Moreover, the authors found tumor DNA in saliva samples
collected after the curative treatment of three patients before
clinical diagnosis of recurrence, but in none of the five patients
without recurrence, showing that tumor DNA in the saliva and
plasma seems to be a potentially valuable biomarker for detec-
tion of HNSCC [86]. These results show a promising utility of
using the detection of mutated DNA in body fluids of HNSCC
patients as a non-invasive test for early detection, diagnosis of
suspicious lesions, disease monitoring, and surveillance. More
studies will be needed to validate these results and the applic-
ability of these tests in HNSCC as it has already been shown for
colorectal, breast, and lung cancers.
5.1. DNA methylation
Perhaps the most extensively evaluated DNA-based markers
were those related to epigenetic changes. One of the main
epigenetic changes that occur in mammals is DNA methyla-
tion, which occurs at the cytosine base of DNA, within CpG
dinucleotides. About 60% of human gene promoters are asso-
ciated with CpG islands and are usually unmethylated in nor-
mal cells. In general, CpG-island methylation is associated with
gene silencing, and DNA methylation can inhibit gene expres-
sion by various mechanisms. Activation of proto-oncogenes
and inactivation of tumor suppressor genes are the major
genetic alterations involved in carcinogenesis.
Several studies have investigated the association between
changes in DNA methylation and tumorigenesis in HNSCC over
the past years. The search for biomarkers to evaluate and
measure the status of normal and pathological processes in
cell biology as well as treatment responses is of paramount
importance. The pursuing of these biomarkers is important for
the identification of individuals in the early stages of cancer and
to stratify patients according to tumor prognosis and response
to therapy profiles. Assuming that cancer results from genetic
and epigenetic alterations, analysis based on gene-methylation
profiles in combination with the pathological diagnosis would
be useful in predicting the behavior of these tumors.
5.1.1. Diagnosis
Saliva is emerging as an alternative diagnostic medium to
detect both local and systemic events through DNA methyla-
tion markers. Puttipanyalears and colleagues [92] observed dif-
ferences in oral rinse samples at ALU sequence methylation
when comparing different groups of tobacco users: methylation
value decreased from normal, light smoker, heavy smoker, and
oral cancer [92]. Many genes have been described as hyper-
methylated in saliva samples from head and neck patients than
96 L. M. R. B. ARANTES ET AL.
in controls. The genes DAPK [5,93–95], DCC [96], E-cadherin
[93,95], EDNRB [96–98], FHIT [95], HOXA9 [99], KIF1A [98],
MGMT [93–95,100], microRNA-137 [101], MINT31 [102],
microRNA-200c-141 [47], NID2 [99], p16 [5,20,93–95,100,102],
PCQAP/MED15 [20], RARβ [95], RASSF1 [5,20,93,102], TIMP3
[20,93], TMEFF2 [95], WIF-1 [95], ZNF14 [103], ZNF160 [103],
and ZNF420 [103] were found to be methylated in saliva from
HNSCC patients when compared to a control group (Table 4).
A promoter methylation panel of DAPK1, p16, and
RASSF1A genes was able to detect tumor presence with an
accuracy of 81% in saliva from HNSCC patients when com-
pared with saliva from healthy nonsmoker controls, with a
sensitivity of 94% and specificity of 87% [5]. In the same
way, a panel including CCNA1, DAPK, DCC, MINT31, and p16
had 34.1% sensitivity and 91.8% specificity in discriminating
HNSCC and healthy individuals [102]. Guerrero-Preston et al.
described a ‘Phase I Biomarker Development Trial’ which
used a panel of two genes: HOXA9 and NID2 94% sensitivity
and 97% specificity, in discriminating OSCC saliva samples
from healthy controls saliva indicating that this may be
useful for early detection [99]. KIF1A and EDNRB hyper-
methylation used as a panel has potential to be developed
as a noninvasive tool for HNSCC detection screening, with
77.4% sensitivity and 93.1% specificity [98]. EDNRB and DCC
methylation (46% sensitivity and 72% specificity) in salivary
rinses compares well to examination by an expert clinician
in clinical risk classification of oral lesions, showing the
viability of those genes in oral premalignancy and malig-
nancy screening in clinical care settings in which expert
clinicians are not available [96]. Also, EDNRB methylation in
salivary rinses was independently associated with histologic
diagnosis of premalignancy and malignancy and may have
potential in classifying patients at risk for oral premalignant
and malignant lesions compared to health controls [97].
Aberrant miR-200c-141 hypermethylation could distinguish
OSCC patient oral rinse from healthy volunteer’s, suggesting
a potential clinical application of this specific miRNA methy-
lation in oral fluids [47]. It is important to note that although
all these panels have high sensitivity and specificity to
detect tumor presence, none of them are being used in
the clinic so far. There is a need for a specific study, combin-
ing all the relevant genes capable of distinguish HNSCC
patients from healthy individual with 100% sensitivity and
specificity. Furthermore, some genes appear more frequently
in saliva studies, such as DAPK, DCC, p16, and EDNRB.
Regarding NPC patients, the use of a panel: DAPK,
E-cadherin, p15, p16, and RASSF1 genes, to distinguish cases
from controls, found that nasopharynx swab had 80% sensi-
tivity and 100% specificity, while mouth and throat rinsing
fluid had 90% sensitivity and 98% specificity, showing its
feasibility to be used in diagnosis [106]. In this same way,
methylated RIZ1 DNA was found in 37% of nasopharyngeal
swabs and 30% in mouth and throat rinsing fluid [107].
In a study subdividing patients by their HPV status, Lim and
colleagues observed significantly higher DNA methylation
levels in saliva collected from HPV-negative HNSCC patients
compared with normal healthy controls. In contrast, a signifi-
cant reduction in DNA methylation was detected in saliva
collected from HPV-positive HNSCC patients compared with
HPV-negative HNSCC patients. In general, DNA methylation
levels were similar or lower in the saliva collected from HPV-
positive HNSCC patients compared with the saliva collected
from normal healthy controls, showing the importance of HPV
status in methylation patterns [104]. The noninvasive nature of
saliva collection facilitates easier patient management, and the
fact that hypermethylation is an early event in oral carcino-
genesis facilitates the use of saliva as minimal invasive bio-
marker for HNSCC early detection.
Biomarker panels in serum showed improved detection
when compared with single markers: HIC1 as a single gene
marker had a sensitivity of 31.4% and a specificity of 92.5%,
while the combination of HIC1 and TIMP3 had 50.0% sensitivity
and 84.5% specificity; and if combine three genes: CCND2, HIC1
and PGP9.5, the panel increases the sensitivity to 52.4% while
the specificity dropped to 81.0% [102]. Another panel including
six genes: GABRB3, IL11, INSR, NOTCH3, NTRK3, and PXN had 77%
sensitivity and 87% specificity, in discriminating methylation
preoperative saliva (OSCC patients) and postoperative saliva
(OSCC patients) plus normal controls, indicating a good panel
for diagnosis [105]. The frequency of aberrant serum methyla-
tion for each marker was 31% for p16, 48% for MGMT, and 18%
for DAPK while no changes on the methylation patterns were
found in the serum of the control group [109]. Regarding p16
gene, plasma from HNSCC patients was 65% methylated, while
was 20% methylated in healthy controls [110]. In another study
also involving hypermethylation of p16 gene, the authors
described that 54.5% of the HNSCC patients had methylation
changes in their serum DNA, while no methylation was found in
the control group [116]. EDNRB hypermethylation was identified
in the serum of 10% of the patients with HNSCC but in none of
the control patients, with 100% specificity but low sensitiv-
ity [108].
Regarding NPC patients, the use of a panel: DAPK, E-cadherin,
and RASSF1 genes, to distinguish cases from controls, found that
nasopharynx plasma had 10% sensitivity and 95% specificity,
probably due to the higher number of cancer cells in body fluid
in direct contact with the primary tumor compared to peripheral
circulation [106]. In the same way, RIZ1 hypermethylation was
found in 23% of NPC plasma [107] and DAPK hypermethylation
in 42% of NPC plasma [111]. It is important to note that methy-
lation of the promoter region of a few genes appear more
frequently in saliva, plasma, and serum studies, such as: DAPK,
DCC, p16, RIZ1, and EDNRB as diagnostic markers.
5.1.2. Prognosis
The prognosis for patients with HNSCC is largely determined
by the stage at presentation. The extent of the tumor, as well
as the presence of lymph node metastases and distant metas-
tases, determines the stage [114]. Disappointingly, survival has
not markedly improved in recent decades because patients
still frequently develop locoregional recurrences, distant
metastases, and second primary tumors [3]. Therefore, finding
biomarkers that are able to identify HNSCC patients at high
risk to develop locoregional recurrences is very important.
Using the full panel (CCNA1, DAPK, DCC, MGMT, MINT31,
p16, and TIMP3), Carvalho and colleagues were able to show
statistically significant associations of pretreatment salivary
rinse methylation with poorer local control and poorer overall
Table 4. Summary of data from relevant methylation markers in body fluids of head and neck tumors.
Tumor
Biomarker name Biomarker behavior site Sample type Sample size Technological approach Application Comments References
RASSF1, p16, RASSF1α and PCQAP were HNSCC Saliva 88 HPV−, 45 HPV+, and 122 MSP PCR Diagnosis Panel which discriminate HPV− and HPV+ [104]
TIMP3, and hypermethylated in HPV-negative controls HNSCC patients from normal healthy
PCQAP/MED15 HNSCC saliva, and p16 and PCQAP control. DNA methylation panel was
were hypomethylated in HPV- higher in HPV− patients than controls
positive HNSCC saliva. (71% sensitivity and 80% specificity).
Conversely, DNA methylation panel was
lower in HPV+ patients than controls
(80% sensitivity and 74% specificity).
p16, DAPK, and Sixty-five percent of tumor HNSCC Saliva 30 tumors and 30 saliva MSP PCR Diagnosis First study to detect hypermethylation in [94]
MGMT hypermethylation were also controls saliva.
hypermethylated in saliva.
ZNF14, ZNF160, Hypermethylation of these genes is HNSCC Saliva Discovery: 44 tumors and Discovery: Illumina Infinium Diagnosis Correlation of the expression and [103]
and ZNF420 detectable in both tumor and 25 mucosa controls. HumanMethylation27 methylation array data allowed for the
saliva (22% sensitivity and 100% Validation: 59 tumors and BeadChips. discovery of candidate genes.
specificity). matched salivary rinse, Validation: qMSP
31 mucosa controls, and
35 salivary rinse controls.
RASSF1A, DAPK1, The panel is able to discriminate HNSCC Saliva 143 patients and 77 MSP PCR Diagnosis [5]
and p16 patients from controls (94% controls
sensitivity and 87% specificity).
HOXA9 and NID2 HOXA9 and NID2 (94% sensitivity and OSCC Saliva 179 patients Discovery: Illumina Infinium Diagnosis [99]
97% specificity). HumanMethylation27
BeadChips.
Validation: qMSP
p16 and MGMT p16 and MGMT were OSCC Saliva 58 patients and 90 controls MSP PCR Diagnosis Hypermethylation of p16 and MGMT [100]
hypermethylated in OSCC genes may be affected by MTHFR
samples. polymorphisms.
GABRB3, IL11, Panel was hypermethylated in OSCC Saliva 13 saliva and tumor (OSCC GoldenGate Methylation Diagnosis 77% sensitivity and 87% specificity in [105]
INSR, NOTCH3, preoperative saliva and patients) and 10 saliva Array (Illumina) discriminating methylation
NTRK3, and hypomethylated in postoperative controls preoperative saliva and postoperative
PXN saliva. saliva.
TIMP3, ECAD, p16, TIMP3, ECAD, p16, MGMT, DAPK, and HNSCC Saliva 90 patients (30 normal MSP PCR Diagnosis/ Aberrant methylation of the panel in [93]
MGMT, DAPK, RASSF1 were methylated in saliva mucosa, 90 tumor, 60 disease monitoring saliva reflects tumor status and is
and RASSF1 and tumor saliva at diagnosis, 22 highly specific of the malignant process
saliva follow-up) (diagnosis).
Methylation of the panel was analyzed in
saliva samples collected during follow-
ups and in 95.5% of the cases; positive
methylation in saliva allowed to detect
abnormality few months before clinical
relapse (disease monitoring).
MicroRNA-137 Hypermethylation of miR-137 HNSCC Oral rinse 99 patients and 99 controls MSP PCR Diagnosis [101]
appears to be a relatively
frequently detected event in oral
rinse of SCCHN patients and may
have future utility as a biomarker
in DNA methylation panels.
ALU ALU hypomethylation in OSCC OSCC Oral rinse 43 patients and 108 COBRA Diagnosis Differences in ALU methylation, when [92]
group. controls comparing different groups of tobacco
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS

users: methylation value decreased

from normal, light smoker, heavy
smoker, and oral cancer.
(Continued )
97
 98

Table 4. (Continued).
Tumor
Biomarker name Biomarker behavior site Sample type Sample size Technological approach Application Comments References
ECAD, TMEFF2, OSCC was detected with 100% OSCC Oral rinse 34 patients and 24 controls MSP PCR Diagnosis [95]
RARβ, and sensitivity and 87.5% specificity
MGMT using the panel.
miR-200c-141 miR-200c-141 methylation could OSCC Oral rinse 15 patients and 7 controls MALDI-TOF Diagnosis [47]
distinguish OSCC patient oral rinse
from healthy volunteers.
EDNRB EDNRB methylation was associated OSCC Salivary rinses 161 patients (21 high-risk qMSP Diagnosis The application of EDNRB salivary rinse [97]
with histologic diagnosis of and 140 low-risk lesions). methylation to define a high-risk
premalignancy and malignancy category of patients within a cohort of
L. M. R. B. ARANTES ET AL.

and may have potential in 161 patients without prior biopsy

classifying patients at risk for oral would have changed the clinical low-
premalignant and malignant risk assessment of five patients with
lesions. mild dysplasia and three patients with
severe dysplasia to high risk based on
methylation analysis of salivary rinses.
KIF1A and EDNRB The panel has 77.4% sensitivity and HNSCC Salivary rinses 101 patients and 61 qMSP Diagnosis KIF1A and EDNRB were methylated in 38 [98]
93.1% specificity. controls and 67.6% of salivary rinses from
HNSCC patients, respectively.
CCNA1, DAPK, The panel has 34.1% sensitivity and HNSCC Salivary rinses 211patients and 527 qMSP Diagnosis [102]
DCC, MINT31, 91.8% specificity. controls
and p16
EDNRB and DCC The panel has 46% sensitivity and HNSCC Salivary rinse 191 patients qMSP Diagnosis [96]
72% specificity.
p15, p16, Nasopharynx swab had 80% NPC M&T rinsing 6 normal NPC, 43 M&T MSP PCR Diagnosis [106]
RASSF1A, sensitivity and 100% specificity, fluid and rinsing fluid, 37 NPC
E-cadherin, and while mouth and throat rinsing NPC swabs swabs, 30 NPC tumor,
DAPK fluid had 90% sensitivity and 98% and 43 peripheral blood
specificity. from control
RIZ1 Methylated RIZ1 DNA was found in NPC M&T rinsing 30 NPC, 30 M&T rinsing MSP PCR Diagnosis Methylated RIZ1 DNA was found also in [107]
37% NPC swabs and 30% M&T fluid and fluid, 30 NPC swabs, 8 23% plasma and 10% buffy coat.
rinsing fluid. NPC swabs normal NP, and 29
peripheral blood from
control
CCND2, HIC1, CCND2, HIC1, and PGP9.5 panel had HNSCC Serum 211 patients and 527 qMSP Diagnosis HIC1 and TIMP3 panel had 50% sensitivity [102]
PGP9.5, and 52.4% and 81% specificity. controls and 84.5% specificity.
TIMP3
EDNRB EDNRB hypermethylation was HNSCC Serum 100 patients and 50 qMSP Diagnosis [108]
identified in the serum of 10% controls
HNSCC patients but in none of the
control patients.
p16, MGMT, and Fifty-five percent of the primary HNSCC Serum 95 patients MSP PCR Diagnosis [109]
DAPK tumors showed hypermethylation
in at least one gene: 27% at p16,
33% MGMT, and 18% DAPK.
p16 Methylated p16 was found in 65% of HNSCC Plasma 20 patients and 24 controls qMSP Diagnosis [110]
the patients and 20% of controls.
RASSF1A, For the panel, NPC plasma had 10% NPC Plasma 6 normal NP, 43 M&T MSP PCR Diagnosis [106]
E-cadherin, and sensitivity and 95% specificity. rinsing fluid, 37 NPC
DAPK swabs, 30 NPC tumor,
and 43 peripheral blood
from control
(Continued )
Table 4. (Continued).
Tumor
Biomarker name Biomarker behavior site Sample type Sample size Technological approach Application Comments References
RIZ1 RIZ1 hypermethylation was found in NPC Plasma 30 NPC, 30 M&T rinsing MSP PCR Diagnosis [107]
23% of NPC plasma. fluid, 30 NPC swabs, 8
normal NP, and 29
peripheral blood from
control
DAPK Hypermethylation of DAPK was NPC Plasma 12 patients MSP PCR Diagnosis [111]
found in both early- and late-
stage NPC.
CCNA1, DAPK, Local disease control and overall HNSCC Saliva rinses 61 patients qMSP Prognosis This finding has potential to influence [112]
DCC, MGMT, survival were significantly lower in treatment and surveillance of HNSCC
MINT31, p16, patients with hypermethylation of patients.
and TIMP3 the panel in saliva rinses.
SHOX2 and SEPT9 Hypermethylation in plasma for the HNSCC Plasma 649 patients and 102 qMSP Prognosis [113]
panel was correlated to a worst control
overall survival (52% sensitivity
and 95% specificity).
TIMP3 Patients with TIMP3 methylation in HNSCC Salivary rinse 200 patients qMSP Disease monitoring This study presents, for the first time, the [114]
samples collected 6 months after detection of TIMP3 promoter
the last curative treatment had hypermethylation in posttreatment
lower local recurrence-free salivary rinse as an independent
survival. prognostic maker for local recurrence-
free survival in patients with HNSCC.
TIMP3 Hypermethylation of TIMP3 had an HNSCC Salivary rinse 197 patients qMSP Disease monitoring [115]
association with local recurrence-
free survival.
p16 p16 methylation was detected at an OSCC Serum 17 patients MSP PCR Disease monitoring Hypermethylation was detected in three [116]
early stage. out of four patients with recurrence.
HOXA2 The higher the Esptein-Barr NPC Plasma 5 patients MALDI-TOF Disease monitoring [117]
virus copy number, the higher the
HOXA2 methylation status and the
higher the MMP-9 in the plasma
of NPC patients.
CDH1, DAPK1, Hypermethylation of at least one of NPC Plasma 41 patients and 43 control qMSP Disease monitoring Hypermethylated CDH1 and DAPK1 were [118]
p15, p16, and the genes was detectable in 71% found in 15% and 23% in plasma from
RASSF1A plasma of NPC patients before NPC recurrent patients, respectively,
treatment. and were not detected in patients of
the control group.
HNSCC: head and neck squamous cell carcinoma; OSCC: oral squamous cell carcinoma; NPC: nasopharyngeal squamous cell carcinoma; M&T: mouth and throat; MSP PCR: methylation-specific polymerase chain reaction; qMSP
PCR: quantitative methylation-specific polymerase chain reaction; COBRA: combined bisulfite restriction analysis; MALDI-TOF: MassArray mass spectrometry platform; EBV: Epstein-Barr virus; HPV: human papillomavirus.
®
Only relevant findings were included in the table.
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS
99
100 L. M. R. B. ARANTES ET AL.
survival [112]. Regarding SHOX2 and SEPT9 genes, aberrant
hypermethylation in plasma from HNSCC patients was corre-
lated to a worst overall survival, indicating that these markers
might be used for prognostic [113].
5.1.3. Disease monitoring
While normal healthy individuals have little cfDNA in their
plasma (~30 ng/mL), a high level of circulating cfDNA
(~180 ng/mL) can be detected in the plasma of cancer
patients. This difference reflects that cfDNA is mainly released
from apoptotic and necrotic tumor cells; and the amount of
cfDNA may be used as a cancer indicator.
Methylation of TIMP3, ECAD, p16, MGMT, DAPK, and
RASSF1A genes was analyzed in saliva samples collected dur-
ing follow-ups and in 95.5% of the cases, and positive methy-
lation in saliva allowed to detect abnormality few months
before clinical relapse. Thus, this study showed that careful
monitoring of patients with positive saliva allowed diagnosis
of tumors when they were still very small and thereby surgi-
cally curable [93]. Nakahara and colleagues found that tumors
which had recurrence also had p16 hypermethylation in OSCC
serum collected 2 months after the surgery (three out of four
cases – one was not possible to evaluate due to tissue necro-
sis), suggesting that circulating DNA might be feasible for
detecting recurrence, because p16 promoter methylation in
the serum was detected at an early stage (TNM Stage I) [116].
Sun and colleagues reported that HNSCC patients with pro-
moter hypermethylation of TIMP3 in salivary rinses have a
poorer local recurrence-free survival than those without hyper-
methylation [115]. Also in another cohort, patients with TIMP3
methylation detected in salivary rinse samples collected
6 months after the last curative treatment had lower local
recurrence-free survival, justifying the use of DNA hyper-
methylation detection in saliva as a tool for identifying and
monitoring HNSCC patients’ subgroups with high risk of devel-
oping local recurrence [114].
As it is easier to obtain plasma samples than biopsies, mea-
surement of aberrant methylated cfDNA in plasma may replace
biopsies in NPC patients [117]. In NPC plasma samples, increased
plasma Epstein-Barr virus (EBV) copy number was correlated
with increased in cell-free HOXA2 hypermethylation. Plasma
EBV DNA and methylated cell-free HOXA2 can be used as bio-
markers for monitoring NPC treatment [117]. Hypermethylated
CDH1 and DAPK1 were found in 15% and 23% in plasma from
NPC recurrent patients, respectively, and were not detected in
patients of the control group (in remission). These results sug-
gested that cell-free circulating methylated gene promoter DNA
is a possibly useful serological marker in assisting in screening of
primary and potentially salvageable local or regional recurrent
NPC [118].
Circulating biomarker evaluations in plasma and serum are
attractive for cancer screening because they are blood-based
tests that are minimally invasive, are relatively low cost, and
demand procedures easily reproducible. The detection of DNA
methylation in body fluids also has the potential to distinguish
high-risk subjects that harbor occult cancers and have a higher
risk for development of solid tumors in a wide variety of
human cancers, including head and neck [108]. Since the
methylated promoter DNA in the circulation reflects the total
body content of methylated cancer cells, we believe that the
quantity of cell-free circulating methylated promoter DNA of
some specific genes in cancer patients should be higher than
in normal controls or patients in remission after treatment.
On the other hand, there are a few studies evaluating
hypermethylation in plasma and serum, mostly because of
the small amount of DNA yield, but new technologies are
overcoming these difficulties, so the number of studies is
increasing. As promoter hypermethylation patterns in indivi-
dual tumors show variation depending on specific altered
molecular pathways, the use of multiple genes will provide
greater applicability and coverage for diverse tumors when
compared with a single gene for general detection [102].
6. Detection of protein-based biomarkers in body
fluids for the management of HNSCC
In a similar manner as transcriptomes identify the whole com-
pendia of cellular transcripts, a proteome represents the whole
collection of intracellular and secreted proteins of a given
tissue or cell population. Protein analysis for cancer diagnosis,
prognosis, and treatment response has been broadly used in
liquid samples, such as saliva, serum, and plasma (Table 5). At
this point, we will focus on protein biomarkers originated at
the tumor site and present in serum or plasma, and saliva,
largely neglecting proteins such as cytokines and growth
factors arising from the general clinical features of cancer.
Recent advances in proteomics have allowed the high-
throughput analysis of liquid biopsies. Besides its nucleic
acid component, saliva contains a large variety of proteinac-
eous analytes. During the last 10 years or so, several proteome
analyses searching for molecular biomarkers for cancer diag-
nosis, tumor progression, metastasis, and treatment response
of premalignant (oral mucositis and leukoplakia) or malignant
HNSCC and OSCC samples were published.
6.1. Diagnosis
Ma and colleagues screened serum samples by matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS) [119]. ELISA validation showed that
decreased GSTP1 concentration was found in patients with
OSCC (0.92 ± 0.11 ng/mL) compared with normal persons
(1.37 ± 0.22 ng/mL) [119].
HPV16 serology was also analyzed as diagnostic marker for
HPV16-driven oropharyngeal squamous cell carcinoma
(OPSCC) [120]. In this work, serum antibodies to E1, E2, E6,
E7, and L1 HPV16 proteins were compared to tumor HPV RNA
status as the gold standard [120]. Moreover, HPV16 E6 sero-
positivity was the best predictor of HPV16-driven OPSCC [120].
Of the 66 HPV-driven OPSCC, 63 were HPV16 E6 seropositive
compared to only 1 (1.8%) among the 54 non-HPV-driven
OPSCC [120]. Outside the oropharynx, HPV16 E6 seropositivity
was reported as less sensitive [120].
Another research showed that serum concentration of
cyclase-associated protein 1 (CAP1) correlated with the
depth of primary tumor invasion and the presence of regional
metastases [121]. In cancer patients, serum level of CAP1 was
Table 5. Summary of data from relevant protein-based markers in body fluids of head and neck tumors.
Biomarker name Biomarker behavior
GSTP1 OSCC samples with lower OSCC
GSTP1 expression than
normal samples.
HPV16 anti-E1, E2, E6, In oropharynx cancer, HPV16 Oropharyngeal
E7, and L1 E6 seropositivity showed squamous cell
an accuracy of 97% and carcinoma
Cohen’s kappa 0.93.
Sensitivity and specificity
were 96% and 98%,
respectively.
Cyclase-associated Serum level of CAP1 in HNSCC
protein 1 cancer samples was lower
than in patients with
laryngeal and
laryngopharyngeal
dysplasia.
Antigens for HPV16, HPV16 E6 seropositivity was HNSCC
HPV6, HPV11, associated with
HPV18, HPV31, oropharyngeal cancer, but
HPV33, HPV45, and not with other cancer
HPV52 sites.
Midkine The sensitivity, specificity, HNSCC
positive predictive value,
negative predictive value,
and accuracy of serum
midkine concentration for
detection of HNSCC were
57.3%, 85.3%, 77.6%,
69.2%, and 72.1%,
respectively.
Gelsolin, fibronectin, Gelsolin, fibronectin, OSCC
angiotensinogen, angiotensinogen, and
and haptoglobin haptoglobin significantly
overexpressed in node-
positive OSCCs versus
node-negative OSCCs
Tumor site Sample type Sample size
Serum Main cohort: 166 OSCC
patients and 120 normal ELISA, and IHC
samples.
Validation cohort (ELISA): 18
OSCC patients and 18
normal samples.
Validation cohort (IHC): 28
OSCC samples.
Serum 214 HNSCC patients:
120 with oropharynx
cancer;
45 with oral cavity cancer;
12 with hypopharynx
cancer;
37 with larynx cancer
Serum 46 HNSCC samples;
12 dysplasia samples;
15 healthy individuals
Serum 180 oral cancers;
135 oropharynx cancers;
247 hypopharynx/larynx
cancers;
300 esophageal cancers;
1599 controls
Serum 103 patients with HNSCC;
116 normal controls
Serum Proteome cohort: 2D liquid
pool with six node-positive
OSCC samples.
Pool with six node-
negative OSCC samples.
Western blot cohort:
16 node-positive OSCC
samples.
12 node-negative OSCC
Technological
approach Application Comments References
MALDI-TOF, Diagnosis Patients with moderately or [119]
poorly pathological
validation differentiation grade
showed significantly lower
GSTP1 expression in the
cancerous tissues than
those with well-
differentiated tumors
(p = 0.041).
Bead-based multiplex Diagnosis Outside the oropharynx, [120]
serology HPV16 E6 seropositivity
was reported as less
sensitive.
ELISA Diagnosis Moreover, patients with [121]
larynx and laryngopharynx
dysplasia had CAP1 serum
levels twofold higher
(49.63 ± 9.54 mg/mL)
than healthy individuals
(25.16 ± 0.24 mg/mL).
Luminex Diagnosis [122]
ELISA Diagnosis and Clinical stages at the time of [123]
prognosis diagnosis were two
patients with stage 0, 13
patients with stage I, 14
patients with stage II, 15
patients with stage III, and
59 patients with stage IV.
Prognosis [124]
chromatography
with tandem mass
spectrometry
(LC-MS/MS)
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS

samples.
(Continued )
101
 102

Table 5. (Continued).
Technological
Biomarker name Biomarker behavior Tumor site Sample type Sample size approach Application Comments References
Alpha-2-HS- Alpha-2-HS-glycoprotein was Hypopharyngeal Plasma Proteome cohort: Proteome by 2D-DIGE/ Diagnosis [125]
glycoprotein found upregulated in squamous cell 8 HSCC samples; MALDI-TOF/TOF MS
HSCC plasma samples. carcinoma (HSCC) 8 healthy donor samples ELISA validation
ELISA cohort:
40 HSCC samples;
40 healthy donor samples
Fibronectin The levels of plasma cellular HNSCC Plasma 127 HNSCC patients; ELISA Diagnosis Authors developed their own [126]
fibronectin, determined by 51 normal subjects antibodies to be used in
ELISA, were found to be ELISA assays.
L. M. R. B. ARANTES ET AL.

increased in Selected patients were at the

gastrointestinal and head preoperative stage,
and neck cancers. undergoing neither
radiotherapy nor
chemotherapy.
Osteopontin (OPN) OPN plasma levels were Laryngeal and Plasma 75 patient samples; ELISA Diagnosis and [127]
significantly higher in hypopharyngeal 20 healthy volunteers prognosis
cancer samples than in squamous cell
control subjects. carcinoma
Osteopontin Tumor location (favoring HNSCC Plasma 27 oral cavity; ELISA Prognosis Samples were dichotomized [128]
oropharynx), relapse type 70 oropharynx; at a cutoff point of
(favoring locoregional), 30 hypopharynx; 450 ng/mL
and OPN (favoring low 13 other anatomical sites
OPN) were independent
prognostic factors for
survival after relapse.
Osteopontin Results indicated no HNSCC Plasma 578 HNSCC patients: ELISA Prognosis [129]
evidence that high plasma 318 oropharynx;
OPN levels were 260 non-oropharynx
associated with an
adverse prognosis in
HNSCC or were predictive
of benefit with hypoxia
targeting therapy
MMP-11 Plasma levels of OSCC Plasma 330 OSCC male patients ELISA Prognosis [130]
endopeptidase MMP-11
were significantly higher
in OSCC patients with
advanced T status, lymph
node metastasis, and
higher TNM stages
MMP-2 and MMP-9 Plasma MMP-9 levels were OSCC Plasma 38 OSCC patients ELISA Prognosis Patients with no evidence of [131]
significantly lower in disease after surgical
responders compared removal of tumor were
with pretreatment levels classified as complete
responders (n = 30), while
patients with locoregional
failure of disease, stable/
progressive disease,
metastasis, or recurrence
were classified as
nonresponders (n = 8)
(Continued )
Table 5. (Continued).
Biomarker name Biomarker behavior
TIMP-1 Plasma levels of TIMP-1 were HNSCC
associated with survival,
with an adjusted hazard
ratio of death of 23.2
TIMP3 Plasma levels of TIMP3 was OSCC
significantly associated
with large tumor in OSCC
patients
Fibrinogen High levels of plasma
fibrinogen were positively
related with growth type,
differentiation, thickness,
and infiltrative growth
ratio
Fibrinogen Plasma fibrinogen levels
were significantly
associated with a
reduction of overall
survival rates
Apolipoprotein A1, All oral leukoplakia cases
alpha amylase, were positive for keratin
cystatins, keratin 10 staining.
10, and lysozyme
precursor
Creatine kinase Authors validated Gal-7 as a OSCC
M-chain, potential screening by
nucleoside Western blot analysis. The
diphosphate results showed a
kinase A, specificity of around 90%
phosphoglycerate and a sensitivity of 80%
mutase, (n = 10), meaning that
glutathione Gal-7 is a good screening
S-transferase, marker for diagnosis of
retinoic acid- OSCC in saliva.
binding protein II,
cofilin, galectin-7,
and C-reactive and
the heat shock
proteins, HSP27,
HSP60, HSP71,
HSP90
Technological
Tumor site Sample type Sample size approach Application Comments References
Plasma 42 oral cancer; ELISA Prognosis [132]
40 laryngeal cancer;
27 oropharyngeal cancer;
23 hypopharyngeal cancer;
4 nasopharyngeal cancer
Plasma 262 OSCC ELISA Prognosis The mean plasma level of [133]
TIMP3 was 2842.87 ±
259.58 pg/mL in small
tumor (≤T2) and 3960.37
± 311.73 pg/mL in large
tumor (>T2)
Oral tongue squamous Plasma 76 patients with clinical Prognosis Multivariate analysis showed [134]
cell carcinoma T1N0M0 or T2N0M0 who that the plasma fibrinogen
had primary surgical level remained an
treatment, from which independent factor for
30 developed cervical disease-free survival after
metastases partial glossectomy
postoperatively and 46 for oral tongue squamous
showed no evidence of cell carcinoma .
disease for 5 years after
surgery.
HNSCC Plasma 206 patients treated with Prognosis Samples were dichotomized [135]
curative intent by at 411 mg/dL of plasma
primary radiotherapy; fibrinogen.
141 patients treated with
curative intent with
postoperative
radiotherapy.
Oral leukoplakia Saliva 10 individuals with oral Proteome by 2DE/ Diagnosis [136]
leukoplakia; MALDI-TOF/TOF
10 nonsmoker control
volunteers
Saliva and OSCC Sample size is confusing. Proteome by 2D-PAGE, Diagnosis Tumor species from two [137]
tissue Abstract and sample followed by MALDI- OSCC patients showed a
preparation section are TOF-MS stage of T3 (1 N0, 6 N2)
conflicting. M0, and from three
patients, a stage of T4 (1
N0, 2 N2) M0. No tumor
cells were detected in the
surrounding mucosal
tissue.
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS
(Continued )
103

Table 5. (Continued).
Biomarker name Biomarker behavior
Annexin 1, zinc finger Authors detected increased HNSCC
protein 28, levels of annexin 1, zinc
regulator finger protein 28,
G-protein 3, regulator G-protein 3,
indoleamine 2,3- indoleamine 2,3-
dioxygenase, OFD1 dioxygenase, OFD1
protein, and protein, and CEP290
CEP290 proteins in most of the
samples from the
malignant cancer patients.
RETN RETN levels in the OSCC
patients were significantly
higher than that in the
healthy or in the oral
potentially malignant
disorders (OPMD) group.
PLUNC, Serum levels of protein
serotransferrin, alpha-1-microglobulin/
transthyretin, and bikunin precursor and
zinc-alpha-2- transthyretin
glycoprotein were higher in serum of
patients before treatment
than
in controls or in patients
after treatment.
Conversely,
haptoglobin exhibited a
higher level of expression
after
radiotherapy.
A1AT, AACT, ANGT, OSCC
ANT3, APOB,
APOH, C1
inactivator, CERU,
CFAH, CFB, CRP,
FA12, FETUA, FIBB,
FINC, HEMO, HEP2,
HPT, HRG, ITIH1,
KNG1, PLMN,
SAA4, SAMP, and
VTNC
104
Technological
Tumor site Sample type Sample size approach Application Comments References
Saliva 25 head and neck cancer Proteome by sodium Diagnosis The subjects with HNSCC [138]
patients and 25 healthy dodecylsulfate- were all heavy smokers (at
volunteers, matching in polyacrylamide gel least one pack per day).
age and gender. electrophoresis
(SDS-PAGE),
followed by MALDI
TOF/TOF mass
L. M. R. B. ARANTES ET AL.
spectrometry
OSCC Saliva Proteome cohort: Proteome by SDS- Diagnosis Clinical staging for proteome [139]
10 healthy volunteers; PAGE, followed by OSCC cohort was: I (2) II
9 individuals with OPMDs; LC-MS/MS. (2), III (2), and IV (4).
10 OSCC patients Clinical staging for ELISA
ELISA cohort: OSCC cohort was: I (23) II
99 healthy volunteers; (16), III (6), and IV (36).
99 individuals with OPMD;
87 OSCC patients
HNSCC Saliva and serum Saliva cohort: Proteome by 2D, Diagnosis The patient set included four [140]
7 male HNSCC patients (6 followed by MALDI- metastatic (N+ or positive)
smokers or former Q-TOF mass and 11 nonmetastatic (N0 or
smokers), before and spectrometry. negative) carcinomas.
after therapy (surgery/
radiotherapy);
10 unmatched healthy
donors (1 female and 9
males, nonsmokers).
Serum cohort:
15 HNSCC patients (1
female and 14 males and
13 smokers or former
smokers);
19 smokers or former
smokers and 7
nonsmokers (1 female and
25 males).
Saliva 61 oral cancer patients; Multiplexed liquid Diagnosis Participants were randomly [141]
58 controls chromatography divided into two
multiple-reaction- independent groups
monitoring mass (clinical set 1 contained 32
spectrometry (LC- OSCC samples and set 2
MRM/MS). contained 29 OSCC
The technique allowed samples; each group
to quantitate 56 contained 29 control
saliva samples).
proteins.
(Continued )
Table 5. (Continued).
Technological
Biomarker name Biomarker behavior Tumor site Sample type Sample size approach Application Comments References
MMP1, KNG1, ANXA2, OSCC Saliva 96 healthy controls; Multiplexed liquid Diagnosis [142]
and HSPA5 103 low-risk OPMDs; chromatography
130 high-risk OPMDs; multiple-reaction-
131 OSCC subjects monitoring mass
spectrometry (LC-
MRM/MS).
C1R, LCN2, SLPI, OSCC Saliva 8 healthy; Multiplexed selected Diagnosis [143]
FAM49B, TAGLN2, 9 OSCC patients who reaction monitoring
CFB, C3, C4B, underwent surgical; (SRM) tandem mass
LRG1, and 13 OSCC patients with spectrometry
SERPINA1 active oral malignant
lesions
Survivin, CEA, and Survivin and CEA levels in OSCC Saliva 26 OSCC patients without ELISA Diagnosis Clinical description well [144]
ErbB2 saliva and local tumor- chemotherapy; presented in Table 1. Most
exfoliated cells of OSCC 10 non-cancer patients patients were SCC grade I
patients were significantly or II.
higher than those in the
control group
CD44 soluble Laryngeal cancer Saliva 21 patients with laryngeal ELISA Diagnosis and [145]
cancer; prognosis
12 controls (adults with
head and neck benign
disease)
CD44 soluble OSCC Saliva 150 cases; ELISA Diagnosis Clinical description well [146]
150 frequency-matched presented in Tables 1
controls and 2.
HNSCC: head and neck squamous cell carcinoma; OSCC: oral squamous cell carcinoma; SCC: squamous cell carcinoma; IHC: immunohistochemistry; MALDI-TOF: matrix-assisted laser desorption ionization-time of flight; 2D-DIGE:
two-dimensional differential gel electrophoresis; TNM: tumor nodal metastasis.
Only relevant findings were included in the table.
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS
105
106 L. M. R. B. ARANTES ET AL.
lower than in patients with laryngeal and laryngopharyngeal
dysplasia [121]. HNSCC patients with regional metastases had
CAP1 levels almost sixfold higher (233.30 ± 94.91 mg/mL) than
patients without metastases [121].
Kreimer and colleagues studied HPV antibodies as prediag-
nostic sera biomarkers for head and neck cancer [122]. The
authors showed that HPV16 E6 seropositivity was present in
prediagnostic samples for 34.8% of patients with oropharyn-
geal cancer and 0.6% of controls (odds ratio, 274; 95% con-
fidence interval, 110–681) but was not associated with other
cancer sites [122].
Tian and colleagues analyzed plasma proteome of 48 hypo-
pharyngeal squamous cell carcinoma (HSCC) and 48 healthy
donors [125]. Among the 26 differentially expressed proteins,
authors validated alpha-2-HS-glycoprotein by ELISA, showing
plasma concentrations of 132.8 ± 20.56 µg/mL and
270.6 ± 38.14 µg/mL (control group and HSCC group, respec-
tively) [125].
Osteopontin (OPN) plasma levels of laryngeal and hypophar-
yngeal squamous cell carcinoma patients showed that OPN
expression was significantly correlated with differentiation and
lymphatic metastasis, where plasma OPN concentrations in
patients (25.696 ± 5.140 ng/mL, n = 75) were significantly
higher than in control subjects (15.222 ± 2.988 ng/mL, n = 20;
p < 0.001) [127]. Another study analyzing plasma samples
showed that cellular fibronectin in plasma at the preoperative
stage, undergoing neither radiotherapy nor chemotherapy, was
found increased in HNSCC patients [126].
In an attempt to identify biomarkers for oral leukoplakia,
Camisasca and colleagues analyzed saliva samples by pro-
teome approach, identifying 22 spots highly abundant in
oral leukoplakias and not detected in the control group,
including apolipoprotein A1, alpha amylase, cystatins, keratin
10, and lysozyme precursor [136]. Using a similar proteomic
approach for OSCC screening, Krapfenbauer and colleagues
identified 25 proteins with altered expression levels in saliva
[137]. Moreover, the authors validated Gal-7 as a potential
screening by Western blot analysis, with a specificity of around
90% and a sensitivity of 80% (n = 10), meaning that Gal-7 may
be a good screening marker for diagnosis of OSCC in saliva
[137]. Saliva proteome analysis detected increased levels of
annexin 1, zinc finger protein 28, regulator G-protein 3, indo-
leamine 2,3-dioxygenase, OFD1 protein, and CEP290 proteins
in most of the samples from the malignant cancer patients
[138]. Another saliva proteome analysis was able to validate
the RETN protein, showing that RETN levels in the OSCC
patients were significantly higher than that in the healthy or
in the oral potentially malignant disorder group [139].
Moreover, the elevated levels of salivary RETN were highly
correlated with late-stage primary tumors, advanced overall
stage, and lymph node metastasis [139]. In a similar approach,
Vidotto and colleagues analyzed the proteomes of saliva and
serum samples of HNSCC patients, detecting overexpression of
PLUNC and zinc-alpha-2-glycoprotein and altered serum levels
of serotransferrin and a modified transthyretin [140].
In a less high-throughput approach, several works have eval-
uated a set of selected proteins for proteomic analysis. In this way,
Chen and colleagues analyzed 56 salivary proteins previously
associated with human cancers as diagnosis biomarkers, using a
multiple reaction monitoring (MRM)-based targeted proteomic
approach incorporating liquid chromatography with mass spec-
trometry detection (LC-MRM/MS) [141]. From this analysis, the
authors showed the levels of 25 proteins with statistically signifi-
cant different protein expression between OSCC samples and
controls, where five of them (CFAH, AFAM, GELS, SAMP, and
VTDB) were used as a diagnosis panel in multivariate analysis of
logistic regression model [141]. Similarly, Yu and colleagues ana-
lyzed a panel of 49 proteins using MRM-MS, in saliva of a cohort
formed by 460 individuals [142]. From the 28 proteins successfully
quantified, the authors developed a four-protein panel consisting
of MMP1, KNG1, ANXA2, and HSPA5, exhibiting high sensitivity
(87.5%) and specificity (80.5%) in distinguishing OSCC samples
from non-OSCC samples [142]. At the same time, Kawahara and
colleagues assayed 14 OSCC biomarker proteins in a set of control
and patient saliva [143]. The authors showed a statistically signifi-
cant higher abundance of the C1R, LCN2, SLPI, FAM49B, TAGLN2,
CFB, C3, C4B, LRG1, and SERPINA1 candidate biomarkers in patient
saliva [143]. In addition, the authors also demonstrated that CFB,
C3, C4B, SERPINA1, and LRG1 were associated with the risk of
developing OSCC [143].
Now, focusing on single biomarker analysis, several
researchers tried in the last decade, to validate individual
proteins in different clinical settings and sample types. In
this way, Li and colleagues evaluated survivin, carcinoembryo-
nic antigen (CEA), and ErbB2 in body fluids (saliva, serum, and
local tumor-exfoliated cells) of OSCC patients for early detec-
tion of oral malignant cancer [144]. In this study, the authors
showed that survivin and CEA levels in saliva and local tumor-
exfoliated cells of OSCC patients were significantly higher than
those in the control group [144].
Another biomarker well studied in saliva is the CD44 pro-
tein. In this way, Allegra and colleagues analyzed CD44 soluble
(CD44sol) levels in saliva of laryngeal carcinoma patients
before and after surgery, reporting that mean salivary levels
of CD44sol in patients were significantly higher than those in
the control group (70.75 ± 33.8 vs. 12.4 ± 8.7 ng/mL), at the
pre-intervention time point [145]. Moreover, in the 3-month
after surgery follow-up, 21 (85.71%) patients showed a reduc-
tion in salivary CD44sol levels [145]. Similarly, Pereira and
colleagues analyzed CD44 in oral rinses, where high-risk indi-
viduals receiving oral cancer prevention interventions were
followed over one year, finding out that CD44sol expression
levels higher than 5.33 ng/mL were highly associated with
case status [146].
6.2. Prognosis
In a high-throughput technological approach, Chai and collea-
gues analyzed the association of the proteomic profile of 282
proteins and the nodal metastasis positivity in serum samples
of OSCC patients, identifying four candidate biomarkers (gel-
solin, fibronectin, angiotensinogen, and haptoglobin) signifi-
cantly overexpressed in node-positive OSCCs versus node-
negative OSCCs [124].
Yamashita and colleagues evaluated the pretreatment
serum midkine expression level as biomarker for HNSCC and
showed that serum midkine concentration was an indepen-
dent prognostic factor, with overexpression of serum midkine

yielding a relative risk of death of 3.77 [123]. Moreover, serum
midkine levels in patients with HNSCC were associated with
malignancy, chemosensitivity, and prognosis [123].
Petrik and colleagues studied the relationship of pretreatment
plasma OPN levels with treatment outcomes in 140 newly diag-
nosed HNSCC patients [128]. Data showed a significant correlation
between OPN and freedom-from-relapse, overall survival, and
event-free survival (EFS) [128]. Moreover, OPN revealed as an
independent prognostic factor for initial tumor control, event-
free survival in those patients achieving tumor control, and post-
relapse survival [128]. In the final model, tumor location (favoring
oropharynx), relapse type (favoring locoregional), and OPN (favor-
ing low OPN) were independent prognostic factors for survival
after relapse [128]. On the other hand, Lim and colleagues studied
the OPN plasma levels in a stage III/IV HNSCC patients treated with
tirapazamine randomized in a phase III trial (TROG 02.02), showing
no evidence that high plasma OPN levels were associated with an
adverse prognosis in HNSCC or were predictive of benefit with
hypoxia targeting therapy [129].
Several matrix metalloproteinase were also studied to ana-
lyze their prognostic behavior in OSCC tumors. In this way,
Hsin and colleagues showed that plasma levels of endopepti-
dase MMP-11 were significantly higher in OSCC patients with
advanced T status, lymph node metastasis, and higher TNM
stages [130]. In addition, the authors called attention to the
fact that, although the levels of MMP-11 were significantly
higher in patients with N2 (16.96 ± 9.93 ng/mL), when com-
pared with patients without nodal metastasis, patients with N1
(17.12 ± 10.05 ng/mL) were not different to N0 patients [130].
Similarly, Patel and colleagues analyzed MMP-2 and MMP-9
plasma expression level in 38 OSCC patients, where 12 of
them were also sampled before initiation of anticancer ther-
apy [131]. Results indicated that plasma MMP-9 levels were
significantly lower in responders compared with pretreatment
levels, while the MMP-9 levels were comparable between pre-
treatment patients and nonresponders [131].
On the other side of the biological effect, metalloproteinase
inhibitors were also studied as prognostic factors. As follows,
Pradhan-Palikhe and colleagues studied the association of
plasma levels of tissue inhibitor of metalloproteinase-1
(TIMP-1) in 136 HNSCC patients, showing that TIMP-1 level
was associated with survival, with an adjusted hazard ratio
(HR) of death of 23.2 [132]. Similarly, Su and colleagues ana-
lyzed the tissue inhibitor of metalloproteinase-3 (TIMP3)
plasma levels in 262 OSCC patients, showing that, among
the 216 betel quid chewers, plasma levels of TIMP3 was sig-
nificantly associated with large tumor in OSCC patients [133].
Peng and colleagues analyzed the association of preopera-
tive plasma fibrinogen levels with clinicopathological para-
meters and disease-free survival in patients with oral tongue
squamous cell carcinoma, showing that high levels of plasma
fibrinogen were positively related with growth type, differen-
tiation, thickness, and infiltrative growth ratio [134]. Univariate
analysis showed that growth type, differentiation, thickness,
and preoperative plasma fibrinogen levels were significantly
correlated with disease-free survival [134]. Patients with a
preoperative plasma fibrinogen level >300 mg/dL were more
likely to develop cervical metastasis [134]. Plasma fibrinogen
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 107
levels were also studied by Selzer and colleagues, in locally
advanced HNSCC patients, reporting that plasma fibrinogen
levels were significantly associated with a reduction of overall
survival rates [135]. Multivariate analysis revealed that fibrino-
gen concentrations were associated with overall survival for
both groups, with HR = 2.5 (postoperative therapy) and
HR = 1.7 (primarily irradiated) [135].
7. Challenges in the path from discovery to clinical
practice
Due to the clinical relevance of biomarkers in clinical decision-
making, it is vital to develop a fluxogram for the process.
Following the identification of candidate biomarkers, the pri-
mary suitability of the candidates is assessed. Comparison
between normal and diseased samples is performed to ensure
that there is a significant difference in abundance between the
normal and diseased statuses. After the assessment, there is a
verification and optimization phase, which consists in a robust
evaluation and verification within a large clinical study followed
by optimization in terms of sensitivity, specificity, and potential
for clinical application or commercialization. The last phase is
the validation and translation, where are performed large-scale
clinical validation trials, in a more generalized population. Then,
validity is assessed in a diagnostic environment and its ability to
detect target disease determines its translational potential.
Finally, the translational pathway usually present a high cost
and unfortunately only a small percentage of the candidate
biomarkers may progress toward a clinical assay [20].
8. Expert commentary
Head and neck tumors affect important sites both anatomically
and functionally. If diagnosed early, treatment is highly cura-
tive; however, patients with advanced tumors will suffer from
toxic organ preservation treatment protocols or extensive sur-
geries, both which may cause a negative impact in anatomical
function, esthetic appearance, and quality of life. Moreover,
advanced tumors present higher rates of recurrence and
worse survival rates. The feasibility of testing molecular markers
in body fluids has the potential to help in early diagnosis,
treatment tailoring, prognosis, and surveillance. The collection
and processing of body fluids such as peripheral blood and
saliva have improved and made feasible to be performed easily
and fast and at low-cost. The technology to test molecular
markers in these fluids, depending on the biomarkers and the
molecules to be evaluated (DNA, RNA, or protein), is available,
but at a wide range of complexity and investment. A qualitative
and quantitative evaluation of single markers such as DNA
epigenetics, mRNA, miRNA, and lncRNA can be reached
through very widespread techniques such as quantitative
qPCR. By qPCR, a low amount of material can yield results
regarding the profile of expression or methylation of these
markers in a rapid, sensitive, and reproducible way. A more
exploratory screening can be achieved using different
approaches such as microarrays, RNA/DNA sequencing, and
the NanoString nCounter technology. Similarly, as transcrip-
tomes identify the whole compendia of cellular transcripts, a
108 L. M. R. B. ARANTES ET AL.
proteome represents the whole collection of intracellular and
secreted proteins of a given tissue or cell population. Protein
analysis for cancer diagnosis, prognosis, and treatment
response has been broadly used in liquid samples, such as
serum, plasma, and saliva. Recent advances in proteomics
have allowed the high-throughput analysis of liquid biopsies.
The most used techniques for proteome analysis are two-
dimensional differential gel electrophoresis (2D-DIGE) and
matrix-assisted laser desorption ionization-time of flight/time
of flight mass spectrometry (MALDI-TOF/TOF MS) followed by a
validation using Western blot and ELISA. It is our belief that, in
the near future, body fluids will be adopted in clinical practice
for diagnosis, prognosis, and monitoring of head and neck
tumors. However, despite all the improvements in sample col-
lection, stabilization, processing and marker retrieval from body
fluids, and the recent advances in technologies to detect and
measure these molecules, no molecular marker has been used
during the management of these tumors. The high heteroge-
neity of these tumors regarding tumor site, biology, and even
the etiology (tobacco versus viral infections), together with the
difficulties in the implementation of an efficient infrastructure
for a reliable use of body fluid-based markers, has hindered its
use in a clinical setting. Therefore, more validation in different
and larger cohorts to evaluate the efficacy of testing molecular
markers in body fluids in head and neck tumors, as well as
improvements in the logistics of doing these tests in a less
complicated and time-consuming approach, seems to be
necessary for this techniques to become routinely used. So
far, the most promising markers are SAT and IL8 mRNA; miR-9
and miR-21 miRNAs; DAPK, p16, MGMT and TIMP3 methylation;
and CD44 protein.
9. Five-year view
The use of liquid biopsy in the clinical practice has been shown
feasible in different settings such as the search for specific
alterations that can help in the selection of patients who will
or will not respond to a specific therapy or in disease monitor-
ing. Even though several studies have shown interesting results
using body fluid-based marker detection in HNSCC, to date,
there is no marker being used in a clinical setting. However,
the use of new techniques such as the digital PCR is making it
possible to detect very rare cancer- or phenotype-specific mole-
cular alterations in a background of normal features, increasing
sensitivity and reproducibility. As soon as this technique
becomes more widespread and the results of successful use
of molecular markers in liquid biopsy in other tumors, mainly
lung, became available, it will surely encourage a deeper assess-
ment of this approach in head and neck cancers.
Key issues
● Head and neck squamous cell carcinoma (HNSCC) encom-
passes tumors arising from several locations that currently
stand as the sixth most common cancer worldwide.
● The main risk factors are tobacco and alcohol consumption,
and, for a subgroup of HNSCCs, infection with high-risk
types of human papillomavirus (HPV).
● Despite several improvements in the treatment of these
tumors in the last decades, overall survival rates have only
improved marginally, mainly due to the advanced clinical
stage at diagnosis and the high rates of treatment failure
associated with this late diagnosis.
● Tissue-based diagnostic strategies used in the clinical set-
ting require the test of materials obtained by invasive
procedures (e.g. biopsies or needle aspirations) commonly
associated with substantial patient discomfort and health
care costs.
● Since molecular alterations precede clinical symptoms and
the possibility of imaging or histopathological-based diag-
nosis, many disorders remain undiagnosed until they have
reached an advanced stage when damage is irreversible,
and treatment is inefficient.
● Molecular-based biomarkers in body fluids (e.g. serum,
plasma, and saliva), namely liquid biopsy, may serve as
highly accurate, reproducible and easy-to-measure tools in
the clinical setting because of the advantages in accessibil-
ity, low invasive procedure, low cost, and the possibility of
multiple sampling for monitoring the disease outcome.
● Several studies have shown the feasibility of using the liquid
biopsy to improve the diagnosis, predict prognosis and facil-
itate surveillance of HNSCC patients. The type of molecule to
be evaluated depends on the end-point use of the test, the
stability of the marker in body fluids and the availability of
materials and equipment for sampling and testing.
● Prospective studies with large cohorts should be performed
in order to test the accuracy, reproducibility, and feasibility
of the most promising markers (e.g. SAT and IL8 mRNA;
miR-9 and miR-21 microRNAs; p16, MGMT, TIMP3 methyla-
tion; and CD44 protein levels) so that they can be reliably
translated into the clinic routine.
Funding
This paper was not funded.
Declaration of interest
AL Carvalho has a National Counsel of Technological and Scientific
Development Scholarship (CNPq). LMRB Arantes is recipient of scholarship
from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP;
grant number 2011/02864-7). The authors have no other relevant affilia-
tions or financial involvement with any organization or entity with a
financial interest in or financial conflict with the subject matter or materi-
als discussed in the manuscript apart from those disclosed. Peer reviewers
on this manuscript have no relevant financial or other relationships to
disclose.
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