Critical Reviews in Oncology/Hematology 110 (2017) 6273

Contents lists available at ScienceDirect

Critical Reviews in Oncology/Hematology

journal homepage: www.elsevier.com/locate/critrevonc

Salivary biomarkers in the diagnosis of breast cancer: A review

Elisa Cancado Porto-Mascarenhas a,b , Daniele Xavier Assad a,c , Hlne Chardin d,e ,
David Gozal f , Graziela De Luca Canto g,h , Ana Carolina Acevedo a ,
Eliete Neves Silva Guerra a,
a
Laboratory of Oral Histopathology, Health Sciences Faculty, University of Braslia, Braslia, Brazil
b
Cettro Centro de Cncer de Braslia, Braslia, DF, Brazil
c
Hospital Srio-Libans, Braslia, DF, Brazil
d
Facult de Chirurgie Dentaire, Universit Paris Descartes Sorbonne Paris Cit, Paris, France
e
ESPCI Paris Tech, Paris, France
f
Department of Pediatrics, The University of Chicago, IL, USA
g
Brazilian Centre for Evidence Based Research, Department of Dentistry, Federal University of Santa Catarina, Florianopolis, SC, Brazil
h
School of Dentistry, Faculty of Medicine and Dentistry, University of Alberta, Canada

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
2. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
2.1. Protocol and registration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
2.2. Study design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
2.3. Study selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
2.4. Charting the data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
2.5. Level of evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.1. Study selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.2. Study characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.3. Level of evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.4. Synthesis of results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Conict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Authors contributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Appendix A. Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

a r t i c l e i n f o a b s t r a c t

Article history: Salivary biomarkers could be helpful to characterize breast cancer. Therefore, this review was
Received 8 April 2016 performed to evaluate the capability of salivary biological markers in the diagnosis and monitor-
Received in revised form ing of breast cancer. Studies were eligible for inclusion if they assessed the potential diagnostic
14 September 2016
value or other discriminatory properties of biological markers in saliva of patients with breast
Accepted 15 December 2016
cancer. The search was performed in six electronic databases (Cochrane, LILACS, PubMed, Sci-
ence Direct, Scopus, Web of Science). In addition the biomarkers were classied according to their
Keywords:
potential clinical application. We identied 567 pertinent studies, of which 13 met the inclu-
Biological markers
Diagnosis
sion criteria. Combined biomarker approaches demonstrated better ability to predict breast cancer
Saliva patients than individual biomarkers. As single biomarker, namely proline, reported great capacity
Breast cancer
Review

Corresponding author at: SQN 205, Bloco H apto 201, Asa Norte, Braslia, DF 70.843-080, Brazil.
E-mail addresses: elieteneves@unb.br, elieteneves.unb@gmail.com (E.N.S. Guerra).
URL: http://mailto:elieteneves@unb.br (E.N.S. Guerra).

http://dx.doi.org/10.1016/j.critrevonc.2016.12.009
1040-8428/ 2016 Elsevier Ireland Ltd. All rights reserved.
 E.C. Porto-Mascarenhas et al. / Critical Reviews in Oncology/Hematology 110 (2017) 6273 63

in both early and late stage breast cancer diagnosis. Taurine showed interesting capability to identify
early breast cancer individuals. Furthermore, valine also demonstrated excellent diagnostic test accuracy
for advanced stages of breast cancer. Only seven studies reported sensitivity and specicity (Zhang et al.,
2010; Streckfus et al., 2000a; Brooks et al., 2008; Cheng et al., 2015; Bigler et al., 2002; Zhong et al.,
2016; Streckfus, 2009), which varied considerably from 50% to 100%, and from 51% to 97%, respectively.
In general, salivary biomarkers identied advanced stages of breast cancer better than early stages. There
is currently limited evidence to conrm the putative implementation of salivary biomarkers as diagnostic
tools for breast cancer. However, current review provides new research directions.
2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction non-invasively collected clinical specimens would be optimal for

Globally, breast cancer is the most frequently diagnosed malig-
nity, corresponding to over a million cases each year (Globocan,
2012). It is also the leading cause of cancer-related deaths in women
worldwide (Siegel et al., 2013). The incidence rates are highest in
North America, Australia/New Zealand, and in Western and North-
ern Europe, and lowest in Asia and sub-Saharan Africa (Jemal et al.,
2011). These geodemographic differences are likely related to soci-
etal changes as a result of industrialization (e.g., changes in fat
consumption, body weight, early menarche and reproductive pat-
terns such as fewer pregnancies and later age at rst birth) (Ahlgren
et al., 2004). In the United States, breast carcinoma accounts for at
least 230,000 cases each year and is responsible for over 40,000
deaths (Siegel et al., 2013). Its mortality rate has been decreasing
since the 1970s (Kohler et al., 2015). This decrease in mortality is
likely due, at least in part, to improved breast cancer screening and
adjuvant therapy (Narod et al., 2015).
Early detection of breast cancer offers the promise of eas-
ier treatment (smaller surgeries, less radiation or chemotherapy)
and improved survival. Conventional screening (physical examina-
tion and mammography) has a less-than-desirable sensitivity and
specicity (Berg et al., 2004). At present, screening mammography
is considered the gold standard for detection of breast cancer; how-
ever, the sensitivity of this test is between 54% and 77% depending
on the type of mammographic procedure (Berg et al., 2004). Thus,
women who undergo a putatively negative annual mammography
may still present with breast cancer. False-positive rates in breast
cancer screening are also a notable limitation, as high callback rates
and dispensable biopsies, increase cost, radiation dose, and patient
apprehension (Drukteinis et al., 2013). A relevant obstacle towards
early recognition of breast carcinoma is the development of meth-
ods that conveniently and accurately identify potentially affected
individuals (Etzioni et al., 2003). The improvement in early detec-
tion of breast cancer is essential for successful patient management
(Arellano et al., 2009). To conrm the diagnosis of breast cancer,
breast biopsies as core biopsy or mammotomy are followed by
a histopathological and immunohistochemistry analysis, although
limitations of these methods have already been reported (Zhang
et al., 2013). The biopsy for diagnosis is invasive and associated in
some cases with patient morbidity. The relative complexity, low
access, and high costs of the gold standard approach employed for
diagnosing the vast majority of breast cancer cases has urged the
eld to search for alternative diagnostic methods to improve early
detection (Zhang et al., 2013).
Technological advancements have beneted biomarker
research to the point where saliva is now recognized as an
excellent diagnostic vehicle that can be collected simply and non-
invasively. Increasing interest has developed in the last decade
on the use of saliva as an adjunct test that enhances conventional
medical assessment approaches to serious systemic diseases
(Streckfus and Bigler, 2005; Streckfus and Bigler, 2002). A sensitive
assay that readily and accurately identies biomarkers using
breast cancer detection and screening (Zhang et al., 2010). As a
diagnostic medium, saliva has several biochemical advantages
when compared with blood. The collection of saliva is safe (i.e., no
needle punctures), noninvasive and relatively simple, and may be
collected repeatedly without discomfort to the patient (Mandel,
1990). In this regard, analyses of salivary biomarkers offer addi-
tional advantages, since they are a ltrated fraction of the blood,
thereby reecting the physiological conditions of the body, such
that salivary samples could be used to monitor clinical status and
predict systemic diseases (Sugimoto et al., 2010; Lawrence, 2002;
Bigler et al., 2009). Saliva-based diagnostics, particularly those
based on metabolomic technologies, are emerging and offer a
promising clinical strategy, characterizing the association between
salivary analyses and a particular disease (Sugimoto et al., 2010).
The capability of salivary biomarkers in the assessment of other
cancers has been studied, e.g. head and neck cancer (Guerra et al.,
2015), but the cumulative experience in the context of breast can-
cer remains unclear. In this context, the aim of this review was
to critically evaluate the potential of salivary biomarkers in breast
cancer diagnosis, and to provide new directions for future studies.
2. Methods
2.1. Protocol and registration
The protocol was registered at the international prospec-
tive register of systematic reviews (PROSPERO) under number
CRD42015024085.
2.2. Study design
A review evaluating the capability of salivary biomarkers for
assessment of breast cancer diagnosis and to monitor treatment of
patients with advanced disease was undertaken.
Articles that focused on salivary biological markers in the diag-
nosis of breast cancer were included. Studies in which salivary
biological media were used as a potential diagnostic media and/or
to monitor adults patients with breast cancer compared with non-
breast cancer controls were also considered.
Studies were excluded for the following reasons: (1) those that
were not primary research articles, including reviews, letters, per-
sonal opinions, book chapters, and conference abstracts; (2) studies
that did not evaluate biomarkers for diagnosis in breast cancer; (3)
those that used different biological media such as blood or body
uids instead of saliva as potential media diagnostics and/or to
monitor adult patients with breast cancer.
2.3. Study selection
Studies to be considered for inclusion were identied using
search strategies for each of the following electronic databases:
Cochrane, LILACS, PubMed, Science Direct, Scopus, Web of Science
64 E.C. Porto-Mascarenhas et al. / Critical Reviews in Oncology/Hematology 110 (2017) 6273
(Supplementary Table S1). A partial grey literature search was also
performed using Google Scholar and references cited in eligible
articles were crosschecked. The searches were performed across
all databases on August 12, 2016 with no time or language restric-
tions. All references were managed by reference manager software
(EndNote, Thomson Reuters, Philadelphia, Penn), and duplicate hits
were removed.
The study selection was completed in two phases. In phase one,
two authors (E.C.P.M. and D.X.A.) independently screened titles
and abstracts identied in all electronic databases. These authors
(E.C.P.M. and D.X.A.) selected articles that appeared to meet the
inclusion criteria based on their abstracts. In phase two, full articles
were evaluated and. E.C.P.M and D.X.A. independently participated.
The reference lists of all included studies were critically assessed
by one reviewer (E.C.P.M.) for any incremental references inadver-
tently omitted during electronic database searches. Disagreements
between the two authors were solved by consensus. When they did
not reach a consensus, a third author (A.C.A.) was involved to make
a nal decision. Any studies that did not fulll the inclusion criteria
were discarded. Final selection was always based on the full-text
of the publication.
2.4. Charting the data
The following information was collected from all included stud-
ies: year of publication, author(s), country, sample size (cases of
breast cancer and non-breast cancer controls), patient age, study
methods, type of biomarkers, class, and main conclusions. If the
required data were not complete, attempts were made to contact
the authors to retrieve the missing information.
One author (E.C.P.M.) collected the required information from
the selected articles; a second author (D.X.A.) crosschecked all data
to conrm the data extraction quality. Any disagreements in either
phase were resolved by discussion with a third author (A.C.A.). A
fourth. reviewer (H.C.) was involved as required, to enable formu-
lation of the nal decision.
2.5. Level of evidence
The selected studies methodology was evaluated indepen-
dently by two authors (E.C.P.M. and D.X.A.) using the critical review
checklist of the revised Quality Assessment of Diagnostic Accu-
racy Studies (QUADAS-2) (Whiting et al., 2011). These authors
scored each item with yes indicating a low bias risk, and no, or
unclear a high bias risk. Disagreements were resolved by a third
reviewer (A.C.A).
3. Results
3.1. Study selection
In phase one of the study, 567 articles were retrieved across the
six electronic databases. After removing the duplicates, 341 articles
remained. A comprehensive evaluation of the titles and abstracts
resulted in the exclusion of 318 studies, leaving 23 articles as poten-
tially relevant. Thirty-eight articles were identied using Google
Scholar, none of which were included. No additional article was
identied or selected from the reference lists of the retained stud-
ies. A full text review was conducted on the 23 articles retrieved
from phase one of the selection. This process led to the exclusion
of 10 studies (Supplementary Table S2), such that only 13 arti-
cles were retained for nal analyses (Zhang et al., 2010; Sugimoto
et al., 2010; Streckfus et al., 2000a; Brooks et al., 2008; Cheng et al.,
2015; Streckfus et al., 2000b; Bigler et al., 2002; Laid et al., 2014;
Zhong et al., 2016; Laid et al., 2016; ztrk et al., 2011; Streckfus
et al., 2006; Streckfus, 2009). A ow chart describing the process of
identication, inclusion, and exclusion of studies is shown in Fig. 1.
3.2. Study characteristics
All selected studies were published in the past sixteen years
(20002016), and conducted in ve different countries: United
States (Zhang et al., 2010; Streckfus et al., 2000a; Brooks et al., 2008;
Streckfus et al., 2000b; Bigler et al., 2002; Streckfus et al., 2006;
Streckfus, 2009), China (Cheng et al., 2015; Zhong et al., 2016),
Morocco (Laid et al., 2014, 2016), Turkey (ztrk et al., 2011) and
Japan (Sugimoto et al., 2010). All were written in English.
All of the studies evaluated salivary biomarkers in adults,
but four studies appraised both saliva and serum (Streckfus
et al., 2000a; Bigler et al., 2002; Laid et al., 2014, 2016). Sam-
ple size ranged from 3 to 49 breast cancer patients, as well as a
similar range of controls. The salivary biomarkers included pro-
teins, mRNA, metabolites and carbohydrate. They were assessed
by different methods, including enzyme-linked immunosorbent
assay (ELISA), enzyme immunoassay (EIA), surface-enhanced laser
desorption/ionization mass spectrometry (SELDI-MS), isotopic
labeling coupled with liquid chromatography tandem mass spec-
trometry (IL-LCMS/MS), capillary electrophoresis mass spectrom-
etry (CE-TOF-MS), reverse transcription-quantitative polymerase
chain reaction (RT-qPCR), quantitative protein immunoblot,
hidrophylic interaction chromatography-ultra-performance liquid
chromatography-mass spectrometry (HILIC-UPLCMS), method
of Lowry, hidrophylic interaction chromatography-electrospray
ionization-mass spectrometry (HILIC-ESIMS) and reversed-phase
liquid chromatography-electrospray ionization-mass spectrome-
try (RPLC-ESIMS). A summary of the descriptive characteristics
of the studies is presented in Table 1.
3.3. Level of evidence
The studies methods were very homogeneous, and all possessed
high methodological quality, although none of the studies fullled
all of the QUADAS-2 methodological quality criteria. For all studies,
except one (Bigler et al., 2002), patient selection (Was a case-control
design avoided?) of the QUADAS 2 criteria was scored as high risk
of bias because each study recruited a group of healthy controls
and a group known to have breast cancer. Additionally, for all stud-
ies, except two (Streckfus et al., 2000b; Laid et al., 2014), index
test of the QUADAS-2 criteria were scored as unclear because no
information about blinding was reported. On average, the selected
studies were considered as having low risk of bias according to the
QUADAS-2 criteria. The complete item list analyzed is presented in
Supplementary Table S3.
3.4. Synthesis of results
Regarding the potential diagnostic biomarkers, CA 15-3 and c-
erb-B-2 were the most frequently assessed biomarkers, evaluated
in three and four of the thirteen studies, respectively (Streckfus
et al., 2000a; Streckfus et al., 2000b; Bigler et al., 2002; Laid et al.,
2014). The most studied class of biomarker was protein, exam-
ined in nine articles (Streckfus et al., 2000a; Brooks et al., 2008;
Streckfus et al., 2000b; Bigler et al., 2002; Laid et al., 2014, 2016;
Streckfus et al., 2006; Streckfus, 2009), followed by metabolites,
analyzed in three articles (Sugimoto et al., 2010; Cheng et al., 2015;
Zhong et al., 2016). The total sample size for this review was 688
adult subjects (319 breast cancer patients and 369 non-breast can-
cer patients). Combined salivary biomarkers were assessed in eight
studies (Zhang et al., 2010; Sugimoto et al., 2010; Brooks et al., 2008;
Cheng et al., 2015; Zhong et al., 2016; Laid et al., 2016; Streckfus
et al., 2006; Streckfus, 2009). Seven studies reported sensitivity and
Table 1
Descriptive characteristics of included studies (n = 13).

Author/Year Country Cases of breast Controls Age (mean in Methods Biomarkers Class Main Conclusions
cancer (non-breast years)
cancer)

Streckfus et al. USA 30 57 50.8 (K) 41.9 ELISA EIA c-erbB-2 CA Protein Salivary c-erbB-2 levels among women with carcinoma of the
(2000a,b) (C) 15-3 breast had a sensitivity (87%) and specicity (65%), with a
cut-point of 110 units/mL in distinguishing BC from the
controls. The c-erbB-2 protein was found to be equal to or to
surpass the ability of CA 15-3 to detect patients with BC.
Streckfus et al. USA 12 15 49.0 (K) 42.4 ELISA EIA c-erbB-2 Protein The mean values for CA 15-3 among the control subjects were
(2000a,b) (C) CA15-3 p53 approximately 4550% lower than the mean value for the
EGFR cancer group. c-erbB-2 was not detected in the saliva or the
cathepsin-D serum of the control subjects. Conversely, the carcinoma group

E.C. Porto-Mascarenhas et al. / Critical Reviews in Oncology/Hematology 110 (2017) 6273

exhibited the presence of c-erb-2. p53 levels were
approximately 25% higher among the control subjects
compared with the cancer group. Saliva and serum levels of
cathepsin-D and EGFR did not appear to be as tumor specic as
CA15-3, c-erbB-2, and p53 when compared across the groups.
The presence of periodontal disease has no effect on marker
levels; the estrous cycle had no effect on salivary marker
levels; and the markers are secreted primarily from the parotid
gland and are ow rate independent.
Bigler et al. USA 25 0 53.6 (K) ELISA EIA c-erbB-2 CA Protein Salivary and serum c-erbB-2 concentrations were able to
(2002) 15-3 detect 87 and 94% of the subjects with cancer, respectively,
demonstrating that erbB-2 protein expression in saliva may be
a very useful diagnostic tool for measuring patient response to
chemotherapy and/or surgical treatment of their disease.
Salivary and serum CA 15-3 marker were able to detect 62 and
75% of the subjects with cancer, respectively. Only salivary
c-erbB-2 protein exhibited a signicant P < 0.001) pre- and
post-operative concentration difference. Serum and salivary
CA 15-3 did not exhibit signicant differences between pre-
and post-operative marker concentrations, but did
demonstrate a decrease in concentrations.
Streckfus et al. USA 3 3 ND (K) ND (C) SELDI-MS Protein peaks Protein The salivary protein spectra produced by cancer subjects had
(2006) higher intensity levels when compared with the saliva of the
healthy control group. The mass peaks with the greater
intensity levels were 18, 113, 170, 228 and 287 k m/z.
Brooks et al. USA 49 49 54.8 (K) 41.4 ELISA VEGF EGF CEA Protein BC patients had higher levels of the three proteins compared to
(2008) (C) controls. The sensitivity and specicity were: 74 and 73% for
VEGF, 78 and 68% for EGF, and 70 and 56% for CEA. The ndings
indicated that the best prediction was from the combination of
salivary VEGF and EGF with a sensitivity of 83%, specicity of
74% and area under the ROC curve (AUC) of 84%.
Streckfus USA 10 40 ND (K) ND (C) IL-LCMS/MS 209 proteins Protein The results of the salivary analyses yielded approximately 209
(2009) proteins in the saliva specimens. These proteins were able to
distinguish between the healthy control, the benign and the
cancer patients. The S100 A7 protein (P31151) demonstrated a
strong presence in the saliva of subjects diagnosed with
carcinoma of the breast.
Sugimoto et al. Japan 30 17 57 (K) 43 (C) CE-TOF-MS 28 metabolites Metabolite 28 metabolites were signicantly different between the
(2010) including patients and healthy controls including taurine and lysine. The
valine, ROC curve analysis of the ability of salivary metabolites to
creatinine, discriminate between samples from patients with breast
taurine, lysine, cancer and controls was 0.973.
arginine, phos-
phocholine,
choline

65
 66
Table 1 (Continued)

Author/Year Country Cases of breast Controls Age (mean in Methods Biomarkers Class Main Conclusions
cancer (non-breast years)
cancer)

Zhang et al. USA 30 63 52.7 (K) 52.2 RT-qPCR, CSTA, TPT1, IGF2BP1, GRM1, mRNA, Protein Transcriptomic and proteomic signatures in saliva can serve as
(2010) (C) Quantitative GRIK1, H6PD, MDM4, S100A8, biomarkers for non-invasive detection of BC. Eight mRNA
protein CA6 biomarkers and one protein biomarker were pre-validated,
immunoblot and in combination, yielded an accuracy of 92% (83% sensitive,
97% specic) on the pre-clinical validation sample set.
ztrk et al. Turkey 15 10 50.8 (K) 46.7 Method of TSA Carbohydrate Salivary sialic acid was signicantly higher in breast cancer
(2011) (C) Lowry patients compared to controls. Sialylation may be increased in
saliva of patients with breast cancer. Increased salivary sialic
acid may be useful as a non-invasive predictive marker for
breast cancer patients.

E.C. Porto-Mascarenhas et al. / Critical Reviews in Oncology/Hematology 110 (2017) 6273

Laid et al. Morocco 29 31 47.24 (K) 43.45 EIA CA 15-3 Protein There was no signicant difference between salivary CA 15-3
(2014) (C) concentration of cases and controls. The correlation between
salivary and serum CA 15-3 concentration was positive and
statistically signicant.
Cheng et al. China 27 28 51.5 (K) ND (C) HILIC- Arg, Orn, Cit, Ala, Met, Gln, Asp, Metabolite Concentrations of 15 SFAAs demonstrated signicant
(2015) UPLCMS Phe, Trp, Pro, Thr, Ser, His, Leu, differences between BC patients at stages III and HC. The AUC
Val, Glu, Lys for SAAF prole index combined Pro, Thr and His was 0.916
(sensitivity 88%, specicity 85%). As single salivary biomarker
Pro proved the highest accuracy in predicting BC stage III
with sensitivity and specicity 70 and 92% respectively (AUC
0.866). The diagnostic potential of 15 SAAFs as early diagnostic
biomarkers for BC was veried.
Laid et al. Morocco 29 31 47.24 (K) 43.45 ELISA IgG anti-MUC1 IgM anti-MUC1 Protein Autoantibodies may play a role in breast cancer screening. It
(2016) (C) IgG anti-HER2 IgM anti-HER2 was observed higher expression of salivary autoantibodies in
BC as compared to healthy women. Salivary IgG anti-HER2 was
not elevated in breast cancer patients comparing the control
group. The expression of autoantibodies according to the
status of the receptor HER2 in mammary tissues did not reveal
any signicance. The highest correlation for screening was the
combination of IgG anti-HER2 and IgG anti-MUC1, with
coefcient of correlation 0.65.
Zhong et al. China 30 25 53.0 (K) ND (C) HILIC-ESIMS, LysoPC (18:2), Palmitic amide, Metabolite Eighteen potential metabolites for diagnosing BC were
(2016) RPLC-ESIMS Phytosphingosine, LysoPC identied. Among the 18 biomarkers, three up-regulated
(18:1), PS (14:1/16:1), LysoPC metabolites, LysoPC (18:1), LysoPC (22:6) and MG
(16:0), Acetylphenylananine, (0:0/14:0/0:0) provided AUC values of 0.920, 0.920 and 0.929
Propionylcholine, LysoPC respectively, showing a high accuracy in predicting BC. The
(22:6), MG (0:0/14:0/0:0), metabolic phenotype of the BC and the HC groups could be
LysoPE (18:2/0:0), PC better distinguished using the combined data from the HILIC
(18:1/16:0), Phenylalanine, and the RPLC columns. In addition, early stage BC patients
Critulline, Histidine, could be differentiated distinctly from those in advanced stage.
N-Acetylneuraminic acid, PE
(22:0/20:4),
4-Hydroxyphenylpyruvic acid

ND = non-determined. BC = breast cancer. K = case. C = control. ELISA = enzyme-linked immune-sorbent assay. EIA = enzyme immunoassay. Biomarkers: c-erbB-2 = human epidermal growth factor receptor 2. CA 153 = cancer
antigen 15-3. p53 = tumor suppressor oncogene protein. EGFR = epidermal growth factor receptor. SELDI-MS = surface-enhanced laser desorption/ionization mass spectrometry. VEGF = vascular endothelial growth factor.
EGF = epidermal growth factor. CEA = carcinoembryonal antigen. IL-LCMS/MS = isotopic labeling coupled with liquid chromatography tandem mass spectrometry. CE-TOF-MS = capillary electrophoresis mass spectrometry.
CSTA = cystatin A (sten A). TPT1 = tumor protein translationally-controlle d1. IGF2BP1 = insulin-like growth factor 2 mRNA binding protein 1. GRM1 = glutamate release or metabotropic glutamate receptor 1. GRIK1 = glutamate
receptor, ionotropic, kainate 1. H6PD = hexose-6-phosphatehexose-6-phosphate dehydrogenase. MDM4 = Mdm4 p53 binding protein homolog. S100A8 = S100 calcium binding protein A8. CA6 = carbonic anhydrase VI. TSA = total
sialic acid. Arg = arginine. Orn = ornithine. Cit = citrulline. Ala = alanine. Met = methionine. Gln = glutamine. Asp = aspartic acid. Phe = phenylalanine. Trp = tryptonphan. Pro = proline. Thr = threonine. Ser = serine. His = histidine.
Leu = leucine. Val = valine. Glu = glutamic acid. Lys = lysine. IgG anti-MUC1 = immunoglobulin G anti-mucine 1. IgM anti-MUC1 = immunoglobulin M anti-mucine 1. IgG anti-HER2 = immunoglobulin G anti-human epidermal growth
factor receptor 2. IgM anti-HER2 = immunoglobulin M anti-human epidermal growth factor receptor 2. LysoPC = lysophosphatidylcoline. PS = phosphatidylserine. MG = monoacylglycerol. LysoPE = lysophosphatidylethanolamine.
PC = phosphatidylcholine. PE = phosphatidylethanolamine. AUC = area under the curve. ROC = receiver operating characteristic. RT-qPCR = reverse transcription-quantitative polymerase chain reaction. HILIC-UPLCMS = hidrophylic
interaction chromatography-ultra-performance liquid chromatographymass spectrometry. SFAA = salivary free amino acid. HC = healthy controls. HILIC-ESIMS = hidrophylic interaction chromatography-electrospray ionization-
mass spectrometry. RPLC-ESIMS = reversed-phase liquid chromatography-electrospray ionization-mass spectrometry.
 E.C. Porto-Mascarenhas et al. / Critical Reviews in Oncology/Hematology 110 (2017) 6273 67

Table 2
Frequency of biomarkers in the included studies.

Biomarkers Number of studies Included studies (Authors and year)

Human epidermal growth factor receptor 2 (c-erbB-2) 3 Streckfus et al. (2000a,b) and Bigler et al. (2002)
Cancer antigen 15-3 (CA 15-3) 4 Streckfus et al. (2000a,b), Bigler et al. (2002) and Laid et al. (2014)
Tumor suppressor oncogene protein (p53) 1 Streckfus et al. (2000a,b)
Epidermal growth factor receptor (EGFR) 1 Streckfus et al. (2000a,b)
Cathepsin-D 1 Streckfus et al. (2000a,b)
Protein peaks 2 Streckfus et al. (2006) and Sugimoto et al. (2010)
Vascular endothelial growth factor (VEGF) 1 Brooks et al. (2008)
Epidermal growth factor (EGF) 1 Brooks et al. (2008)
Carcinoembryonal antigen (CEA) 1 Brooks et al. (2008)
209 proteins 1 Streckfus (2009)
Metabolites including Phosphocholine, choline 1 Sugimoto et al. (2010)
CSTA 1 Zhang et al. (2010)
TPT1 1 Zhang et al. (2010)
IGF2BP1 1 Zhang et al. (2010)
GRM1 1 Zhang et al. (2010)
GRIK1 1 Zhang et al. (2010)
H6PD 1 Zhang et al. (2010)
MDM4 1 Zhang et al. (2010)
S100A8 1 Zhang et al. (2010)
CA6 1 Zhang et al. (2010)
Total sialic acid (TSA) 1 ztrk et al. (2011)
IgG anti-MUC1 1 Laid et al. (2016)
IgM anti-MUC1 1 Laid et al. (2016)
IgG anti-HER2 1 Laid et al. (2016)
IgM anti-HER2 1 Laid et al. (2016)
Arginine 2 Cheng et al. (2015) and Sugimoto et al. (2010)
Ornithine 2 Cheng et al. (2015) and Sugimoto et al. (2010)
Citrulline 3 Cheng et al. (2015), Zhong et al. (2016) and Sugimoto et al. (2010)
Alanine 2 Cheng et al. (2015) and Sugimoto et al. (2010)
Methionine 1 Cheng et al. (2015)
Glutamine 2 Cheng et al. (2015) and Sugimoto et al. (2010)
Aspartic acid 2 Cheng et al. (2015) and Sugimoto et al. (2010)
Phenylananine 3 Cheng et al. (2015), Zhong et al. (2016) and Sugimoto et al. (2010)
Tryptonphan 2 Cheng et al. (2015) and Sugimoto et al. (2010)
Proline 2 Cheng et al. (2015) and Sugimoto et al. (2010)
Threonine 2 Cheng et al. (2015) and Sugimoto et al. (2010)
Serine 2 Cheng et al. (2015) and Sugimoto et al. (2010)
Histidine 2 Cheng et al. (2015) and Sugimoto et al. (2010)
Leucine 1 Cheng et al. (2015)
Valine 2 Cheng et al. (2015) and Sugimoto et al. (2010)
Glutamic acid 2 Cheng et al. (2015) and Sugimoto et al. (2010)
Lysine 2 Cheng et al. (2015) and Sugimoto et al. (2010)
LysoPC (18:2) 1 Zhong et al. (2016)
Palmitic amide 1 Zhong et al. (2016)
Phytosphingosine 1 Zhong et al. (2016)
Lysophosphatidylcoline LysoPC (18:1) 1 Zhong et al. (2016)
Phosphatidylserine (PS) (14:1/16:1) 1 Zhong et al. (2016)
Lysophosphatidylcoline (LysoPC) (16:0) 1 Zhong et al. (2016)
Acetylphenylananine 1 Zhong et al. (2016)
Propionylcholine 1 Zhong et al. (2016)
Lysophosphatidylcoline (LysoPC) (22:6) 1 Zhong et al. (2016)
Monoacylglycerol (MG) (0:0/14:0/0:0) 1 Zhong et al. (2016)
Lysophosphatidylethanolamine (LysoPE) (18:2/0:0) 1 Zhong et al. (2016)
Phosphatidylcholine (PC) (18:1/16:0) 1 Zhong et al. (2016)
Histidine, N-Acetylneuraminic acid 1 Zhong et al. (2016)
Phosphatidylethanolamine (PE) (22:0/20:4) 1 Zhong et al. (2016)
4-Hydroxyphenylpyruvic acid 1 Zhong et al. (2016)

Table 3
Frequency of the class of biomarkers in the included studies.

Class of biomarker Number of studies Included studies (Authors and

year)

Protein 9 Streckfus et al. (2000a,b), Bigler

et al. (2002), Streckfus et al.
(2006), Brooks et al. (2008),
Streckfus (2009), Zhang et al.
(2010), Laid et al. (2014) and
Laid et al. (2016)
Metabolite 3 Sugimoto et al. (2010), Cheng
et al. (2015) and Zhong et al.
(2016)
mRNA 1 Zhong et al. (2016)
Carbohydrate 1 ztrk et al. (2011)
68 E.C. Porto-Mascarenhas et al. / Critical Reviews in Oncology/Hematology 110 (2017) 6273

COCHRANE LILACS PUBMED SCIENCE DIRECT SCOPUS WEB OF SCIENCE

n= 12 n= 0 n= 156 n= 19 n= 222 n= 158

Records idened through database searching

Idencaon

(n=567)

Records aer duplicates removed

(n= 341)

GOOGLE SCHOLAR
(n=38)

Records screened from databases

(n =23)
Screening

Records screened
from GOOGLE
SCHOLAR Addional studies idened from reference lists
(n = 0) (n =0)

Full-text arcles assessed for eligibility

(n =23)

Full arcles excluded with reasons (n =10)

Eligibility

(1) Reviews, leers, editorials, book

chapters, personal opinions and
conference abstracts (n=5);
(2) Studies that did not evaluate
biomarkers for diagnosis in breast cancer
(n=5);
(3) Dierent biological media, such as
blood or body uids instead of saliva
were used as a potenal media
diagnoscs and/or monitoring adults
paents with breast cancer (n=0);
Included

Studies included in qualitave and quantave synthesis

(n=13)

Fig. 1. Flow Diagram of Literature Search and Selection Criteria adapted from PRISMA (Moher et al., 2010).

specicity (Zhang et al., 2010; Streckfus et al., 2000a; Brooks et al.,
2008; Cheng et al., 2015; Bigler et al., 2002; Zhong et al., 2016;
Streckfus, 2009), which varied considerably from 50% to 100%, and
from 51% to 97%, respectively. The classication for each study is
presented in Table 1. All potential salivary biomarkers for breast
cancer diagnosis and monitoring are presented in Tables 2 and 3.
4. Discussion
This review investigated the cumulative evidence on the accu-
racy of salivary biomarkers in the diagnosis of breast cancer. To the
best of our knowledge, this study is the rst review concerning the
diagnostic capability of salivary biomarkers in breast cancer, exclu-
sively. The gold standard for breast cancer screening and diagnosis,
mammography and breast biopsy, respectively, carries signicant
limitations such as high cost and morbidity, therefore we need
to develop new procedures less inconvenient that ensure a safe
technique which could be used in all stages of breast cancer: screen-
ing, diagnosis, treatment and monitoring for metastasis prediction
(Maric et al., 2011; Porika et al., 2011). This critical need to nd an
ideal biomarker for breast cancer may account for the large number
of studies that have contemplated this theme since 1999.
A tumor biomarker is any biological material provided by the
cancer cell that may be detected and used as indicator of tumor
status or of outcomes in the context of therapeutic interventions
(Passiglia et al., 2015). Biomarkers are designed for varied goals,
comprising to diagnose, to screening and to predict diseases, as well
as to guide individual treatment decisions (Sargent et al., 2005).
There are ve distinct stages to conceptualize the development
of a tumor biomarker. They are: Stage 1: exploratory preclinical
 E.C. Porto-Mascarenhas et al. / Critical Reviews in Oncology/Hematology 110 (2017) 6273
studies with objective to identify potential biomarkers; Phase 2:
Development of testing able to identify/quantify the biomarker
(genes, proteins) in clinical samples, testing their specicity and
interlaboratory reproducibility; Phase 3: longitudinal retrospective
study (assess correlation capacity); Phase 4: prospective screen-
ing studies; Phase 5: Control Studies (assess clinical impact) (Pepe
et al., 2001; Baker et al., 2006). It may be identied and measured
by one or more assays or tests (Hayes, 2015). Clinical diagnosis
of a disease using saliva, especially cancer, probably requires the
analysis of a combinatorial prole of biomarkers to achieve an
acceptable level of sensitivity and specicity. There is no single
mechanism, pathway, or molecule responsible for carcinogenesis
(Glazer et al., 2009), and the diagnostic value for malignancies is
limited if only one tumor marker is examined. Over the past 50
years, the pace of salivary research has accelerated with the advent
of new techniques that illuminate the biochemical and physico-
chemical properties of saliva (Schipper et al., 2007). There is clearly
no consensus regarding which biomarkers possess the best diag-
nostic value, which assays constitute the most accurate type of
biomarker (i.e., proteins, nucleic acids, or metabolites), or which
is the best detection method to use.
CA15-3 is a large transmembrane glycoprotein, which is fre-
quently overexpressed, aberrantly glycosylated in cancer, and
appears to play a role in cell adhesion (Duffy et al., 2000). It is the
most widely used serum marker to detect recurrent breast cancer
and monitor treatment of patients with advanced disease. Agha-
Hosseini et al. (2009) assessed the association between serum and
saliva levels of CA15-3 and compared them between women with
and without breast cancer. The authors found that the salivary and
serum levels of CA15-3 were signicantly higher in cancer patients
and in stage 2 breast cancer, with a signicant positive correla-
tion between serum and saliva CA15-3 concentrations, allowing a
potential use of salivary CA15-3 in the initial detection of breast
cancer in women (Agha-Hosseini et al., 2009). The study by Streck-
fus et al. showed that CA 15-3 levels were able to detect 65% of the
malignant cases (Streckfus et al., 2000a). This value correspond to
value reported by Bigler et al., whose ndings showed sensitivity
of 62% to identify malignant cases (Bigler et al., 2002). In contrast,
Laid reported that there was no signicant difference between sali-
vary CA 15-3 concentrations of breast cancer and healthy patients.
While the correlation between salivary and serum CA 15-3 concen-
trations was positive and statistically signicant in this study (Laid
et al., 2014). The reasons that could explain these different conclu-
sions could be: methodological differences, the sample size, assays
that are not standardized or lack reproducibility, and equivocal
statistical analyses (McShane et al., 2005).
Previous studies have demonstrated that saliva and serum
from breast cancer patients exhibited elevated levels of c-erbB-
2, carcinoma antigen 15-3 (CA 15-3), EGFR, Cathepsin-D and p53,
suggesting that there is communication between the breast tumor
and the salivary gland (Streckfus et al., 2000b; Bigler et al., 2002).
Streckfus et al. demonstrated that the salivary levels of c-erbB-2, CA
15-3 and p53 in the breast cancer patients were notably higher than
the salivary levels of control group subjects, suggesting that these
biomarkers have potential use in initial diagnosis and/or follow-
up for the detection of breast cancer (Streckfus et al., 2000b). In
accordance with the aforementioned study, Bigler et al. illustrated
that salivary and serum c-erbB-2 concentrations were capable to
detect 87 and 94% of the subjects with cancer, respectively (Bigler
et al., 2002). The proto-oncogene HER-2/neu (c-erbB-2) encodes a
transmembrane tyrosine kinase growth factor receptor, which is
a component of a four-member family of closely related growth
factor receptors, including EGFR or HER-1; HER-2; HER-3 and HER-
4. Both HER-2/neu gene amplication and protein overexpression
are detected in about 20% of breast carcinomas and predict a more
aggressive clinical course (Gonzalez-Angulo et al., 2009; Curigliano
69
et al., 2009; Eroles et al., 2012). Streckfus and Bigler showed that
salivary Her-2 exhibits a signicant difference between pre- and
post-therapy values and could be used for postoperative follow-up
evaluation and indications of patient relapse (Streckfus and Bigler,
2005).
Epidermal growth factor (EGF) is a regulatory growth factor pro-
tein, implicated in tumorigenesis and responsible for a variety of
tissue growth and repair associated with poor prognosis and high
degree of invasiveness in breast cancer (Mandel, 1990; Hirata and
Orth, 1979). Based on this line of thought Navarro et al. (1997)
demonstrated that EGF concentrations were higher in the saliva of
women with primary or recurrent breast cancer in comparison with
women lacking a malignancy. The highest concentrations of EGF
were found in the local recurrence subgroup, suggesting a poten-
tial use for this marker in the post-operative follow-up of diagnosed
cancer patients. Angiogenesis plays a critical role in breast tumor
growth and metastasis and elevated angiogenic factors were found
in saliva of patients with cancer (Folkman, 2002). Based on these
data, Brooks et al. (2008) cogitated that a prole of angiogenic and
tumor markers in saliva, such as vascular endothelial growth factor
(VEGF), EGF, and carcinoembryonic antigen (CEA) could be comple-
mentary to the current methods used for breast cancer diagnosis. It
was observed that the levels of the above proteins in the saliva are
elevated in breast cancer patients in comparison to normal controls,
especially when the biomarkers were assessed in combination.
Still regarding protein as a frequently studied class of biomarker,
many researchers had an interest in autoantibodies against tumor
biomarkers. Mucin1 (MUC1) is a transmembrane glycoprotein
formed by two subunits that is glycosylated and overexpressed
in breast tumor in over 90% and plays a pivotal role in progres-
sion of the disease, once MUC1 promotes growth, metastasis, and
resistance to drugs in cancer (Nath and Murkherjee, 2014). It is con-
sidered one of the most specic and validated antigens in breast
cancer patients (Kufe, 2009).
Many data corroborated that the immune system often responds
against tumor cells earlier, thus, autoantibodies produced against
tumor biomarkers may provide a helpful approach in cancer screen-
ing. Prolonged survival for breast cancer patients with elevated
levels of tumor-specic immunoglobulins has been described in
various studies (von Mensdorff-Pouilly et al., 2000; Gourevitch
et al., 1995). As advantages, autoantibodies are present in high
concentration in blood, persist long periods in patients circula-
tion (Murphy et al., 2012) and could be evaluated in saliva which
could afford noninvasive alternative for breast cancer screening
(Arif et al., 2014).
Fremd et al. evaluated the serum level of anti-MUC1 antibod-
ies in regard to tumor biologic parameters as well as the impact
on survival in breast cancer patients. The data conrmed the IgG
anti-MUC1 as an independent prognostic marker in breast can-
cer patients joining high anti-MUC1 IgG levels with statistically
signicantly improved overall survival (Fremd et al., 2016).
Based on that, a very recent report (Laid et al., 2016) investi-
gated if serum and salivary autoantibodies isotypes, IgG and IgM,
against HER2 and MUC1 play a role in breast cancer screening.
According to the results, higher expression of all serum and sali-
vary autoantibodies in breast cancer comparing to healthy women
were found, suggesting that those autoantibodies may play a role
in breast cancer screening. In addition, saliva detection of antibod-
ies of both anti-MUC1 and anti-HER2 in association may be more
interesting than serum. The authors also analyzed the correlation
between the different isotypes of antibodies to evaluate the pos-
sibility to use both immunoglobulin anti-MUC1 and anti-HER2 in
breast cancer screening. The data revealed that the highest corre-
lation for screening was the combination of salivary IgG anti-HER2
and salivary IgG anti-MUC1, with coefcient of correlation 0.65.
The only marker that was not signicantly higher in both patients
70 E.C. Porto-Mascarenhas et al. / Critical Reviews in Oncology/Hematology 110 (2017) 6273
and healthy donors was salivary IgG anti-HER2. These ndings are
endorsed by previous studies (Chapman et al., 2007; Tang et al.,
2010; Lu et al., 2012; Isla Larrain et al., 2013; Evans et al., 2014).
Saliva has also been assayed for protein prole as a breast can-
cer marker. Streckfus et al. (2006) conducted a study to assess if
salivary protein proles were possibly altered in the presence of
carcinoma of the breast. The study was able to identify a number
of proteins that were higher in intensity among the cancer subjects
when compared with controls. The mass peaks with the greater
intensity levels were at the 18, 113, 170, 228 and 287 k m/z ranges
using SELDI analyses.
Zhang et al. (2010) tested the hypothesis that there are cancer
discriminatory biomarkers in saliva for breast cancer using de novo
discovery and validation approaches. It was the rst paper of this
kind, and no other study has engaged in a de novo biomarker dis-
covery approach in saliva for breast cancer detection. In that study,
a case-control discovery and independent preclinical validations
were conducted to evaluate the performance and translational util-
ities of salivary transcriptomic and proteomic biomarkers for breast
cancer detection (Zhang et al., 2010). This review showed that the
panel of 9 combined biomarkers with 8 mRNA biomarkers and
one protein (CSTA + TPT1 + IGF2BP1 + GRM1 + GRIK1 + H6PD + MDM4-
+ S100A8 + CA6) exhibited excellent diagnostic test accuracy (DTA)
for breast cancer diagnosis (Zhang et al., 2010). Interestingly, CA6,
which was validated in this study, was also discovered in a previous
proteomic proling study using saliva samples from non-invasive
breast cancer patients (DCIS) (Streckfus et al., 2008). In addition,
the study of 9 combined biomarkers showed excellent diagnostic
capability result with an accuracy of 92% (Zhang et al., 2010).
Schwarzenbach and Pantel analyzed the circulating
plasma/serum DNA in patients with breast cancer (Schwarzenbach
and Pante, 2015). Cell-free DNA or circulating cfDNA is DNA that
originates in the bloodstream. This can be caught as a biological
sample such as serum or blood for disease examination and is
appropriate for a range of research applications such as real-time
PCR, digital PCR, and next-generation sequencing (Schwarzenbach
and Pante, 2015). Gevensleben et al. (2013) showed that the
plasma cfDNA copy number ratio of HER2 to a reference gene had
an accuracy of 0.92 and that 64% of patients with HER2-amplied
cancer and 94% of patients with HER2-nonamplied cancer dis-
played positive and negative predictive values of 70% and 92%,
respectively. The ndings of this review conrmed this statement
and demonstrated that c-erbB-2 is a single salivary biomarker with
excellent DTA values in the assessment of breast cancer (Streckfus
et al., 2000a).
Metabolites are a biomarker class extensively studied in saliva of
breast cancer patients with screening and diagnosis purpose. Cheng
et al. (2015) observed that proline proved to be a good biomarker
for early stage of breast cancer diagnosis. Proline is a nonessential
amino acid, which is converted to hydroxyproline and hydroxyly-
sine, the two important components of collagen. In our review,
proline emerged as one of the promising salivary biomarkers as it
meets some of the expectation criteria allowing for early diagnosis
of breast cancer. Another amino acid that deserves to be highlighted
is valine, due to excellent diagnostic test accuracy for advanced
stages of breast cancer. Fang et al. (2007) and Sugimoto et al. (2010)
detected a trend for decreasing levels of valine in pancreatic and
breast cancer tissue, respectively.
Previous metabolomics studies have shown that some metabo-
lites can appropriately differentiate breast cancer patients from
normal controls (Budczies et al., 2015; Tenori et al., 2015). Sugimoto
et al. (2010) designed the rst study to extensively investigate
salivary metabolites and to describe metabolic proles specic to
oral, breast and pancreatic cancer. In relation to breast cancer, 28
metabolites were signicantly different between the patients and
healthy control. The ROC curve analysis of the ability of salivary
metabolites to discriminate between samples from patients with
breast cancer and controls was 0.973. This study revealed that sev-
eral metabolites in breast cancer patients yielded a statistically
signicant difference between breast cancer and healthy controls,
including taurine and lysine. Taurine is the most abundant free
amino acid in humans and plays an essential role in several bio-
logical processes, including the regulation of oxidative stress. In the
cancer context, oxidative stress is a major factor responsible for tis-
sue damage. It is known that taurine defends cells against oxidative
injury and it was observed that taurine reaches high concentrations
in tissues exposed to elevated levels of oxidants (Marcienkievcz
and Kontny, 2014) Serum taurine level was investigated as an early
marker for breast cancer in Egyptian patients (El Agouza et al.,
2011). The results showed steadily lower serum taurine level in
breast cancer group than the high risk and control group. It is sug-
gested that assessment of taurine level in serum of patients with
high risk for breast cancer are usefulness in the early diagnosis
of the disease. In line with several studies, these metabolites are
promising biomarkers for breast cancer screening (Sugimoto et al.,
2010).
In this review, one study (ztrk et al., 2011) analyzed the cor-
relation between salivary total sialic acid and breast cancer. Sialic
acid, the end moieties of the carbohydrate chains are biologically
important and essential for functions of glycoconjugates and they
are altered in cancer patients. Increased sialylation of cell surface
glycoconjugates is among the key molecular changes associated
with malignant transformation and cancer progression (Shah et al.,
2008). Until 2011, there was no data about salivary sialic acid in
breast cancer. ztrk was the rst to report signicantly increased
salivary sialic acid levels in breast cancer patients compared with
the healthy control group (ztrk et al., 2011). This result con-
tributed to establish the clinical importance of salivary sialic acid
as a diagnostic marker.
In the years 2000 and 2007, the Clinical Practice Guidelines on
breast cancer tumor markers by the American Society of Clinical
Oncology (ASCO) indicated that there was insufcient evidence to
recommend CEA, CA 153 or Cancer Antigen 2729 (CA 2729) for
screening and diagnosis (Bast et al., 2001). In 2015, ASCO endorsed
that the tumor serum biomarkers CEA, CA 153, and CA 2729
may be used as complementary assessments to collaborate to deci-
sions concerning therapy for metastatic breast cancer (Poznak et al.,
2015). No mention by this panel has yet been made however
regarding the use of salivary biomarkers.
In summary, wide range of salivary biomarkers have thus far
been reported to present a good potential to detect breast cancer
patients versus healthy patients. These ndings suggest that the
majority of the single biomarkers may estimate how likely patients
with the disease can be correctly ruled out, which could contribute
to improvements in the radiologic diagnosis of breast cancer; how-
ever, single salivary-based biomarkers cannot estimate the true
negatives cases. Thus, biomarkers need to be highly specic for can-
cer, and the use of several of such biomarkers as a signature will
likely be necessary for future implementation of an effective overall
screening program with high sensitivity and specicity to poten-
tially enhance our capacity to detect breast cancer early (Pepe et al.,
2001). We consider that the salivary transcriptome, proteome and
metabolome are part of a novel potentially promising diagnostic
alphabet that has been only preliminarily explored in the discov-
ery of breast cancer biomarkers and deserves incremental research
efforts toward such goal.
Some methodological limitations of this review should be con-
sidered. Firstly, only 13 articles met the inclusion criteria out of 341
identied citations. Secondly, few studies met the criteria for inclu-
sion and few studies evaluated the same biomarker, which reduces
the robustness of the ndings. Furthermore, all included studies,
except one (Bigler et al., 2002) have a case-control design in which
 E.C. Porto-Mascarenhas et al. / Critical Reviews in Oncology/Hematology 110 (2017) 6273
they are pre-identied before performing the index test. Although
a common procedure among DTA studies, this design may mislead
index test performance estimates, and result in overestimation of
diagnostic accuracy (Rutjes et al., 2005).
5. Conclusion
This review indicates a paucity of studies on salivary
biological markers in the diagnosis of breast cancer. The
biomarkers analyzed could be used with the purpose of early
diagnosis and to monitor advanced disease. The combin-
ing of CSTA + TPT1 + IGF2BP1 + GRM1 + GRIK1 + H6PD + MDM4-
+ S100A8 + CA6 presented excellent DTA, accurately distinguish-
ing patients with breast cancer from controls. Taurine seems
encouraging with regard to identify early breast cancer, but the
date are incipient and further research is required to discover
and validate salivary biomarkers able to diagnose patients with
breast cancer at an early stage. Proline may also be a promising
single biomarker for future studies, in early stages breast cancer
diagnoses. Valine demonstrated excellent DTA for advanced stages
of breast cancer. The increased salivary total sialic acid may be a
useful predictive marker. The summary ndings from this analysis
show the growing importance of metabolites in salivary biomarker
discovery and can guide future research.
However, to date the evidence is too restricted to assertively
endorse the use of salivary biomarkers as diagnostic tools for breast
cancer.
Conict of interest
The authors declare that they have no competing interests.
Authors contributions
ECPM, DXA and ENSG designed the protocol and the search strat-
egy, performed study selection, data extraction, and synthesis, and
drafted the manuscript. GDLC and ACA participated in interpreta-
tion of the results and helped to draft the manuscript. DG and HC
conceived the study, participated in its design and coordination,
participated in study selection and interpretation of the results,
and helped to draft the manuscript. All authors read and approved
the nal manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.critrevonc.2016.
12.009.
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Biographies
Elisa Cancado Porto-Mascarenhas, MD, Oral Histopathology Laboratory,
Health Sciences Faculty, University of Braslia, Braslia, Brazil. Medical oncologist
of Cettro, Centro de Cncer de Braslia. And general practitioner of Hospital de Base
do Distrito Federal.
Graziela De Luca Canto, DDS, MSc, PhD, Department of Dentistry, Brazilian Cen-
tre for Evidence-Based Research, Federal University of Santa Catarina, Florianopolis,
SC, Brazil. And School of Dentistry, Faculty of Medicine and Dentistry, University of
Alberta, Canada. Dr. De Luca Canto completed a postdoctoral fellowship in Evidence
Based Dentistry with Dr Carlos Flores-Mir at University of Alberta. She understands
evidence-based theories and advanced research designs, uses advanced statistical
methods, and apply clinical research integration in planning and conducting future
clinical research. She is procient in organizing and conducting systematic reviews
as well as meta-analysis and can use systematic reviews associated software like
Endnote, Refworks, and Revman. She has 40 SRs published, 10 in press and 13 in
consideration.
Eliete Neves Silva Guerra, DDS, MSc, PhD, Oral Histopathology Laboratory,
Health Sciences Faculty, University of Braslia, Braslia, Brazil. Dr. Guerra works
closely as a research team under the guidance of Dr. De Luca Canto, one of the
Evidence-based practices experts in South America. During 2014-15, this team has
 E.C. Porto-Mascarenhas et al. / Critical Reviews in Oncology/Hematology 110 (2017) 6273 73

published or has in press 5 SRs about diagnosis and management in Head and neck
cancer. They have demonstrated consistent methodological skills to conduct and
guide Systematic Reviews. Dr. Guerra completed a postdoctoral fellowship in 2010 in
Molecular Biology with Dr. Edward Odell at Kings College, London. She understands
evidence-based theories and advanced research designs, uses advanced statistical
methods, and apply pre-clinical and clinical research integration in planning and
conducting future pre-clinical and clinical research. She is procient in system-
atic reviews as well as meta-analysis and can use systematic reviews associated
software like Endnote and Revman. She has 11 SRs published, 2 in press and 6 in
consideration.