-
Review
Early Diagnostic Biomarkers for EsophagealAdenocarcinoma—The
Current State of Play
Alok Kishorkumar Shah1, Nicholas A. Saunders1, Andrew P.
Barbour2, and Michelle M. Hill1
AbstractEsophageal adenocarcinoma (EAC) is one of the two most
common types of esophageal cancer with
alarming increase in incidence and very poor prognosis. Aiming
to detect EAC early, currently high-risk
patients are monitored using an endoscopic-biopsy approach.
However, this approach is prone to sampling
error and interobserver variability. Diagnostic tissue
biomarkers related to genomic and cell-cycle abnormal-
ities have shown promising results, although with current
technology these tests are difficult to implement in
the screening of high-risk patients for early neoplastic
changes. Differential miRNA profiles and aberrant
protein glycosylation in tissue samples have been reported to
improve performance of existing tissue-based
diagnostic biomarkers. In contrast to tissue biomarkers,
circulating biomarkers are more amenable to
population-screening strategies, due to the ease and low cost of
testing. Studies have already shown altered
circulating glycans and DNA methylation in BE/EAC, whereas
disease-associated changes in circulating
miRNA remain to be determined. Future research should focus on
identification and validation of these
circulating biomarkers in large-scale trials to develop in vitro
diagnostic tools to screen population at risk for
EAC development. Cancer Epidemiol Biomarkers Prev; 22(7);
1185–209. �2013 AACR.
IntroductionAfterheart disease, cancer is the second leading
cause of
death globally. Four major cancer sites account for half ofthe
cancer-related mortalities: lung, colorectal, prostatein men, and
breast in women. In past 2 decades, a steadydecrease in deaths of
these 4 major site malignancies ledto an overall decrease in
cancer-related death rates inmenand women (1). In contrast, the
incidence of esophagealadenocarcinoma (EAC) is increasing faster
than any othercancer type. EAC togetherwith esophageal squamous
cellcarcinoma (ESCC) is the eighth most-common cancer byprevalence
and sixth most-common cause of cancer-relat-ed death globally (2).
In 1970s, the incidence of EACrepresented less than 5% of total
esophageal cancer, anda majority of esophageal cancer cases
diagnosed wereESCC. Over a period of 3 decades, EAC incidences
havebeen increasing continuously, especially inwestern coun-tries
among Caucasians. Now almost half of the esoph-ageal malignancy
cases diagnosed are EAC (3, 4). EACand ESCC show marked differences
in their geographicspread. EAC is more common in developed
countriessuch as the United Kingdom (8 in 100,000 individuals;
ref. 5), Australia, and the United States. Within
Europe,southern Europe has the highest EAC incidence (5). Onthe
other side, ESCC is the most common type of esoph-ageal cancer
amongdevelopingAsian countries (6). Racialdisparity also occurs
between the 2 types of esophagealcancer. ESCC is more prevalent
among Blacks, whereasEAC is at least twice as common in Whites as
comparedwith other ethnic groups (7, 8). Once diagnosed,
Blackpatients showed poorer overall survival than Whites(9, 10).
Taken together, strong genetic and environmentalfactors relating to
ethnicity and geographic distributionseem to be playing critical
roles in the incidence of esoph-ageal cancer. Studies also suggest
possible links betweensocioeconomic status and the prevalence of
esophagealcancer phenotype (6).
Risk FactorsIn themajority of cases, EAC is diagnosed at a late
stage,
leading to a poor 5-year survival of less than 15% (11).Hence,
recent research for EAC has focused on under-standing risk factors
and the identification of early diag-nostic biomarkers.
Esophageal cancer is unlikely to develop in individualsyounger
than 40 years of age; however, after that theincidence increases
significantly with each decade of life(9). Changing lifestyle and
food habits are primarilyresponsible for the dramatic epidemiologic
changes inEAC as described in recent reviews (11–13). Known EACrisk
factors include accumulation of visceral fat in theabdomen (14),
male gender, high intake of dietary fat andcholesterol with low
intake of fruits and vegetables (15),tobacco smoking (16),
reduction in Helicobacter pylori
Authors' Affiliations: 1The University of Queensland Diamantina
Institute;and 2School of Medicine, The University of Queensland,
Woolloongabba,Queensland, Australia
Corresponding Author: Michelle M. Hill, The University of
QueenslandDiamantina Institute, Level 5, Translational Research
Institute, 37 KentStreet, Woolloongabba, QLD 4102. Phone:
61-7-3443-7049; Fax: 61-7-3443-6966; E-mail: [email protected]
doi: 10.1158/1055-9965.EPI-12-1415
�2013 American Association for Cancer Research.
CancerEpidemiology,
Biomarkers& Prevention
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-
infections (17), and Barrett’s esophagus (BE), a metaplas-tic
change to the esophageal lining. Individuals withBarrett’s
esophagus carry almost 30 to 125 times morerisk for EAC
development, and 0.5% to 1% of patientswith Barrett’s esophagus are
estimated to develop EACeach year (18). Barrett’s esophagus is
characterized byreplacement of normal stratified squamous
epitheliumwith metaplastic columnar epithelium and is consideredto
be a successful adaptation of the distal esophagus inresponse to
chronic gastroesophageal reflux disorder(GERD; ref. 19).
GERD is a very common condition in the western pop-ulation with
around 20% reporting weekly symptoms ofheartburn and acid
regurgitation (20). Refluxate-contain-ing bile acid, along with
gastric acid, is considered to bemore harmful, leading to
inflammation, ulceration, Bar-rett’s esophagus, and ultimately EAC.
Development ofBarrett’s esophagus is a slow process and
distinctivemucus-secreting goblet cell formation can take 5 to
10years (21, 22). Typically, EAC develops through
metapla-sia–dysplasia–carcinoma sequence involving genetic
andepigenetic modifications, leading to uncontrolled
cellproliferation, and is characterized by the presence
ofintestinal metaplasiawith low-grade (LGD) to high-gradedysplasia
(HGD), which eventuallymay progress to inva-sive carcinoma
(20).
Current Diagnosis ScenarioTo detect pathologic changes leading
to EAC develop-
ment before onset of disease, current clinical practiceinvolves
endoscopic screening of patients with high-riskGERD and to
characterize the degree of dysplasia inbiopsy samples collected
during endoscopy (23, 24).Enrollment of patients into an endoscopic
screening pro-grammay be facilitated by a patient questionnaire of
self-evaluated symptoms/complications (25, 26). Onceenrolled into
the screening program, a patient undergoesendoscopy-biopsy every 3
months to 2 years dependingon the degree of dysplasia, during which
4 quadrantbiopsy samples are taken every 1 to 2 cm and evaluatedfor
histologic changes by expert pathologists (23, 24). As asignificant
number of patients histologically diagnosedwith HGD develop EAC,
endoscopic mucosal ablation oresophageal resection (esophagectomy)
are options to stopfurther disease progression in those high-risk
patients(27, 28). Significantly improved survival is observed
inpatients diagnosed at an early stage during surveillanceendoscopy
program as compared with symptomaticallydiagnosed EAC (29–32).
Although current screeningmethodology shows prom-ise, outcome of
endoscopy-biopsy in many cases is non-reproducible due to
interobserver variability and sam-pling error (28, 33).
Furthermore, histologic dysplasticchanges may be patchy and present
heterogeneously inthe tissue sample. This makes the diagnosis
challenging,especially in the early stages of transition to LGD
(28, 34).In up to 40%of patients, invasive cancer has been found
inresected tissue despite negative endoscopic examination
for the malignancy (35). Moreover, false-positive resultsalso
occur, meaning despite intramucosal carcinoma in abiopsy, the
subsequently resected tissue has no signs ofcarcinoma (28). These
evidence suggest dysplasia gradingis an imperfect measure of cancer
risk.
Despite extensive screening with currently availabletechniques,
more than 80% of EACs are diagnosed with-out any prior diagnosis of
Barrett’s esophagus or GERD(36, 37). According to an estimate, more
than 80% ofBarrett’s esophagus cases are undiagnosed and
thereforeare not getting the benefit of the screening program
(38).On the other hand, a large proportion of patients under-going
routine biopsy screening do not progress to EAC(13). These suggest
inability of current methodologies inscreening population to detect
high-risk patients and todistinguish between disease progressors
from nonpro-gressors. In addition, the screening procedure is not
verycost-effective (39). To overcome these challenges, adjunctuse
of biomarker has been proposed to stratify the riskassociated with
EAC development.
Biomarkers in EACAccording to United States’ NIH, a biomarker is
"a
characteristic that is objectively measured and evaluatedas an
indicator of normal biologic processes, pathogenicprocesses, or
pharmacologic responses to a therapeuticintervention (40)."
In transit from intestinal metaplasia to LGD to HGD toEAC, cells
acquire abilities to become self-sufficient forgrowth, evade
apoptosis, proliferate uncontrollably, pro-mote angiogenesis,
invade underlined epithelium, andstart to metastasize. These
changes are accompanied withhistologic changes in tissue
architecture, genomic insta-bility, development of tumor
microenvironment, modu-lation of immune response, and are therefore
reflected inbody fluids (serum/plasma/mucus/urine) or tissue
sam-ples and differentiate in terms of their
genome/prote-ome/metabolome profile (41). Thus, a biomarker can
befrom any of these sources and reflect underlying patho-logic or
homeostatic changes. Table 1 summarizes differ-ent classes of
biomarkers proposed for BE/EAC.
National Cancer Institute Early Detection ResearchNetwork (EDRN)
guidelines outline biomarker discoveryanddevelopment to a 5-phase
process summarized below(42) and depicted in Fig. 1.
Phase I—Preclinical exploratory study: it comparesnormal versus
cancer samples (body fluids/tissue)using technologies such as
genomics, microarrayexpression, proteomics, immunohistochemistry,
orimmunoblotting to detect significant changes
inproteins/genes/metabolites between the groups.
Phase II—Clinical assay development and validation: it isaimed
at developing a clinical assay using a minimallyinvasive sample
collection method. The assay is meantto be robust, reproducible,
and suitable for storedclinical samples to be used in later phases
ofdevelopment. At the end of this phase, one should
Shah et al.
Cancer Epidemiol Biomarkers Prev; 22(7) July 2013 Cancer
Epidemiology, Biomarkers & Prevention1186
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expect high specificity and sensitivity for the assay.However,
it remains to be determined how early thebiomarker can predict the
disease.
Phase III—Retrospective longitudinal repository studies:the
assay is applied on prospectively collected storedsamples to
determine the ability of the biomarker todetect the disease before
clinical presentation. If so, thencriteria for positive screening
is determined for futureuse.
Phase IV—Prospective screening: the test is prospectivelyapplied
to real population to detect the extent andcharacteristic of
disease detected by the biomarker. Thisphase gives positive
predictive value for the test andgives an idea about feasibility
for last phase of controltrials.
Phase V—Cancer control studies: it comprises large-scaleclinical
trial to determine the impact of new screeningprocess on the
disease burden in the community.
With respect to EAC, none of the biomarkers, includinghigh-grade
dysplasia, have been evaluated in phase V,whereas very feware
evaluated in phase III and IV. Figure1 summarizes proposed EAC
biomarkers and how wellthey are characterized in the process of
biomarker dis-covery. The following sections will discuss some of
theclasses of BE/EAC biomarkers.
Genomic InstabilityMany groups have studied genomic instability
induced
by aneuploidy, tetraploidy, DNAmethylation, allelic lossand
shown some predictive power for these changes. Arole for
hypermethylation in the promoter regions oftumor-suppressor genes
during the development of EAChas also been well established. Table
2 summarizes DNAmethylation changes associated with
metaplasia–dyspla-sia–carcinoma development. In the majority of
patients,methylation changes are acquired very early during
EACdevelopment, hence these alterations could be used as anearly
diagnostic biomarker. Apart from discriminatingpatients at
different stages of EAC development, DNAmethylation signatures may
be useful as predictors forprogression from Barrett’s esophagus to
EAC (43, 44) andfor response to chemotherapy and survival in
patientswith EAC (45, 46).
Although the individual genomic abnormality has thepotential to
diagnose disease at different stages, bestresults are obtained when
they are used in combination(47–49). LOH at chromosome 9p and 17p
locus are con-sidered to be early events during Barrett’s
esophaguspathogenesis (50). If present with other
chromosomalalterations such as aneuploidy and tetraploidy,
itincreases the 10-year risk for development of EAC from12% to
approximately 80% (51). However, with the cur-rent flow cytometry
technology, it is technically verychallenging for clinical
laboratories to assess these geno-mic biomarkers in the samples,
which limits widespreaduse of these biomarkers in the clinic.
Alternatively, genomic alterations canbedetected at theprotein
level using immunohistochemistry. One of themost common and
earliest genomic abnormality occurs atchromosome 17p, which codes
for tumor-suppressor p53protein. Loss of p53 protein expression in
tissue samplescorrelates with disease progression (52). However, as
p53expression only reflects alterations at one particular gene,it
has lower predictive value as comparedwith techniquesmonitoring
multiple genomic abnormalities. Further-more, sensitivity drops as
mutations or deletions at geno-mic level may not necessarily be
detected at the proteinlevel (53).
In line with the genomic abnormalities described ear-lier,
single-nucleotide polymorphism (SNP)–based geno-typing can also
stratify cancer risk in patients with Bar-rett’s esophagus. As
summarized in Table 3, in the past
None
Phase V: Cancer control studies
Bio
mar
ker d
isco
very
and
dev
elop
men
t
Phase IV: Prospective screening
Phase III: Retrospective longitudinal repository studies
Phase I and II: Preclinical exploration, clinical assay
development and validation
High-gradedysplasis
DNA methylation, LOH,ploidy, p53 loss, cyclin D1
PCNA, Ki-67, EGFR, COX-2,miRNA, cMYC, HER2, NF-κB, Bcl-2,VEGF,
E-cadherin, p16 abnormalities,
β-catenin, glycoproteins, etc.
Figure 1. Summary of current BE/EAC Biomarkers with respect to
EDRNclinical phase of development.
Table 1. Comprehensive summary of differentclasses of BE/EAC
biomarkers
Biomarker class Ref.
Tissue biomarkersGenomic abnormalities(ploidy and LOH)
(47–51)
DNA methylation Refer to Table 2SNPs/expression array studies
Refer to Table 3
Inflammatory markersCOX-2 (69, 72–77)NF-kB (78–81)Cytokines (67,
79, 81–86)MMPs (87–93)Cell-cycle abnormalities (94, 95, 101)miRNA
Refer to Table 4Glycosylation changes (121, 123–125)
Circulatory biomarkersDNA methylation changes (130–132)Glycan
alterations (135–138)Metabolic profiling (142–145)
Biomarkers for Esophageal Adenocarcinoma
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Tab
le2.
Sum
maryof
hypermethy
latedge
nesduringBE/EAC
dev
elop
men
t Num
ber
(%)o
fsa
mplessh
owinghy
permethy
lationorstud
yfind
ings
Gen
eLo
cation
Func
tion
Metho
dNorm
alBE
LGD
HGD
EAC
Ref.
p16
(orCDKN2A
orINK4A
)9p
21Cyc
lin-dep
ende
ntkina
seinhibitor
Methy
latio
n-sp
ecificPCR
5/9(56%
)14
/18(77%
)—
—18
/21(85%
)(146
)
Methy
latio
n-se
nsitive
sing
le-stran
dco
nformationan
alysis
0/10
(0%)
4/12
(33%
)3/11
(27%
)3/10
(30%
)18
/22(82%
)(147
)
Methy
latio
n-sp
ecificPCR
0/17
(0%)
14/47(30%
)9/27
(32%
)10
/18(56%
)22
/41(54%
)(148
)Methy
latio
n-sp
ecificPCR
2/64
(3%)
14/93(15%
)—
—34
/76(45%
)(149
)Methy
latio
n-sp
ecificPCR
—3/10
(30%
)—
—5/11
(45%
)(150
)Methy
latio
n-sp
ecificPCR
—27
/41(66
%)
21/45(47%
)17
/21(81%
)65
/107
(61%
)(151
)Methy
latio
n-sp
ecificPCR
0%1/15
(7%)
4/20
(20%
)12
/20(60%
)8/15
(53%
)(152
)Methy
latio
n-sp
ecificPCR
Sep
aratelyde
term
ined
exon
1an
d2methy
latio
n.Five
of16
(31%
)exo
n-1,
8/16
(50%
)exo
n2in
EAC
patient
samples
show
edhy
permethy
latio
n.Exo
n2
methy
latio
nco
rrelates
with
stag
eof
thetumor
(P¼
0.01
)
(153
)
O6-M
ethy
lgua
nine
-10
q26
DNArepa
irMethy
light
tech
nique
2/10
(20%
)8/13
(62%
)—
—84
/132
(64%
)(154
)DNA Methy
ltran
sferase
(orMGMT)
Methy
latio
n-sp
ecificPCR
6/29
(21%
)24
/27(89%
)13
/13(100
%)
37/47(79%
)(155
)
APC
5q21
-q22
Wnt/b-caten
insign
aling
Methy
latio
n-sp
ecificPCR
0/17
(0%)
24/48(50%
)14
/28(50%
)14
/18(78%
)20
/32(63%
)(148
)
Methy
latio
n-se
nsitive
sing
le-stran
dco
nformation
analysis
and
methy
latio
n-se
nsitive
dot
blotas
say
0/16
(0%)
11/11(100
%)
——
20/21(95%
)(156
)Eight
of14
histolog
ically
norm
alga
stric
muc
osaad
jace
ntto
EAC
show
edsign
ifica
ntly
differen
tmethy
latio
nof
APC
promoter.
(157
)
GSTM
21p
13.3
Antioxidan
tsan
dprotec
tionag
ains
tDNAdam
age
Bisulfite
pyrose
quen
cing
(sam
ple
size
:EAC-
100,
BE-11,
dys
plasia-
11,n
ormal
esop
hage
al/
gastric
muc
osa-37
)
-
Tab
le2.
Sum
maryof
hypermethy
latedge
nesduringBE/EAC
dev
elop
men
t(Con
t'd)
Num
ber
(%)ofsa
mplessh
owinghy
permethy
lationorstud
yfind
ings
Gen
eLo
cation
Func
tion
Metho
dNorm
alBE
LGD
HGD
EAC
Ref.
Tach
ykinin-1
(TAC1)
7q21
-22
Smoo
thmus
cle
contractility,e
pith
elial
iontran
sport,
vasc
ular
permea
bility
andim
mun
efunc
tion
Methy
latio
n-sp
ecificPCR
5/67
(7.5%)
38/60(63.3%
)12
/19(63.2%
)11
/21(52.4%
)41
/67(61.2%
)(162
)
Rep
rimo
2q23
Reg
ulates
p53
-med
iatedce
ll-cy
cle
arrest
inG2-pha
se
Methy
latio
n-sp
ecificPCR
0/19
(0%
)9/25
(36%
)—
7/11
(64%
)47
/75(63%
)(163
)
E-C
adhe
rin16
q22
.1Caþ
2-dep
enden
tintercellularad
hesion
andmaintains
norm
altis
suearch
itecture
Methy
latio
n-sp
ecificPCR
0/4(0%)
——
—26
/31(84%
)(164
)
SOCS-3
17q25
.3Inhibits
cytokine
sign
aling
Methy
latio
n-sp
ecificPCR
0%4/30
(13%
)6/27
(22%
)20
/29(69%
)14
/19(74%
)(165
)
SOCS-1
16p13
.13
0%0/30
(0%
)1/27
(4%
)6/29
(21%
)8/19
(42%
)
Sec
retedfrizzled
-related
proteins(SFR
P)
SFR
P1
8p11
.21
Wnt
antago
nist
Methy
latio
n-sp
ecificPCR
7/28
(25%
)30
/37(81%
)—
—37
/40(93%
)(166
)SFR
P2
4q31
.318
/28(64%
)33
/37(89%
)—
—33
/40(83%
)SFR
P1
8p11
.21
Methy
latio
n-se
nsitive
sing
le-stran
dco
nformationan
alysis
andmethy
latio
n-se
nsitive
dot
blotas
say
1/12
(8%
)6/6(100
%)
——
23/24(96%
)(156
)
SFR
P2
4q31
.311
/15(73%
)6/6(100
%)
——
19/25(76%
)SFR
P4
7p14
.1Methy
latio
n-sp
ecificPCR
9/28
(32%
)29
/37(78%
)—
—29
/40(73%
)(166
)SFR
P5
10q24
.16/28
(21%
)27
/37(73%
)—
—34
/40(85%
)Plako
philin-1
(PKP1)
1q32
Cella
dhe
sion
and
intrac
ellularsign
aling
Methy
latio
n-sp
ecificPCR
5/55
(9.1%)
5/39
(12.8%
)—
1/4(25%
)20
/60(33.3)
(167
)
GATA
-48p
23.1-p22
Tran
scrip
tionfactor
andregu
late
cell
differen
tiatio
n
Methy
latio
n-sp
ecificPCR
0/17
(0%
)—
——
31/44(71%
)(168
)
GATA
-520
q13
.33
0/17
(0%
)—
——
24/44(55%
)CDH13
(orH-
cadhe
rinor
T-ca
dhe
rin)
16q24
Cella
dhe
sion
Methy
latio
n-sp
ecificPCR
0/66
(0%
)42
/60(70%
)15
/19(78.9%
)16
/21(76.2)
51/67(76.1%
)(169
)
(Con
tinue
don
thefollo
wingpag
e)
Biomarkers for Esophageal Adenocarcinoma
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on June 7, 2021. © 2013 American Association for Cancer
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Published OnlineFirst April 10, 2013; DOI:
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http://cebp.aacrjournals.org/
-
Tab
le2.
Sum
maryof
hypermethy
latedge
nesduringBE/EAC
dev
elop
men
t(Con
t'd)
Num
ber
(%)o
fsa
mplessh
owinghy
permethy
lationorstud
yfind
ings
Gen
eLo
cation
Func
tion
Metho
dNorm
alBE
LGD
HGD
EAC
Ref.
NELL
-1(nel-like1)
11p15
Tumor
suppress
orMethy
latio
n-sp
ecificPCR
0/66
(0%)
28/60(46.7%
)8/19
(42.1%
)13
/21(61.9%
)32
/67(47.8%
)(170
)Eye
sAbse
nt4
6q23
Apop
tosismod
ulator
Methy
latio
n-sp
ecificPCR
2/58
(3%)
27/35(77%
)—
—33
/40(83%
)(171
)A-kinasean
choring
protein
12(or
Gravinor
AKAP12
)
6q24
-25.2
Cell-sign
aling,
adhe
sion
,mito
gene
sis,
and
differen
tiatio
n
Methy
latio
n-sp
ecificPCR
0/66
(0%)
29/60(48.3%
)10
/19(52.6%
)11
/21(52.4%
)35
/67(52.2)
(172
)
Vim
entin
10p13
Cytos
keletonprotein
Methy
latio
n-sp
ecificPCR
0/9(0%
)10
/11(91%
)—
5/5(100
%)
21/26(81%
)(173
)RUNX3
1p36
Tran
scrip
tionfactor
Methy
latio
n-sp
ecificPCR
1/63
(2%)
23/93(25%
)—
—37
/77(48%
)(149
)HPP1
19pter-p13
.1Tu
mor
suppress
or2/64
(3%)
41/93(44%
)—
—55
/77(71%
)3-OST-2
16p12
Sulfotran
sferas
een
zyme
1/57
(2%)
47/60(78%
)—
—28
/73(38%
)
Wnt
inhibitory
factor-1
(WIF-1)
12q14
.3Wnt
antago
nist
Methy
latio
n-sp
ecificPCR
81%
ofpa
tientswith
Barrett's
esop
hagu
ssu
fferingfrom
EAC
show
edhy
permethy
latedWIF-1
asco
mpared
with
20%
ofpa
tientswith
Barrett's
esop
hagu
swith
outEAC
(174
)
CHFR
(che
ckpoint
with
forkhe
adasso
ciated
and
ringfing
er)
12q24
Mito
sisch
eckpoint
protein
Bisulfite
pyros
eque
ncing
EAC
samples31
%(18/58
)sho
wed
sign
ifica
ntly
high
erCHFR
promoter
methy
latio
nas
compared
with
norm
alsa
mples(P
¼0.01
).(175
)
Metallothione
in3
(orMT3
)16
q13
Metal
homeo
stas
isan
dprotec
tion
agains
tDNAda
mag
e
Bisulfite
pyros
eque
ncing
(sam
ple
size
:normal-33,
BE-5,E
AC-78)
Iden
tified
2region
s(R2an
dR3)
ofCpG
nucleo
tides
,which
show
edsign
ifica
ntly
high
ermethy
latio
nin
EACas
compared
with
norm
alep
ithelium
(FDR<0.00
1).
Increa
sedDNAmethy
latio
nof
MT3
promoter
R2co
rrelates
with
adva
nced
tumor
stag
e(P
¼0.00
5)an
dlymphno
demetas
tasis(P
¼0.03
).DNAmethy
latio
nof
MT3
promoter
R3co
rrelates
with
tumor
stag
ing(P
¼0.03
)but
notwith
lymph
nodestatus
(P¼
0.4).
(176
)
Methy
lationmarke
rpan
el
Sam
ple
size
Metho
dFind
ings
Ref.
EAC-35un
dergo
ing
chem
orad
iotherap
yMethy
latio
n-sp
ecificPCR
Com
bine
dmea
nof
promoter
methy
latio
nof
p16
,Rep
rimo,
p57
,p73
,RUNX-3,
CHFR
,MGMT,
TIMP-3,a
ndHPP1was
lower
inpatientswho
resp
onded
toch
emorad
iotherap
y(13/35
)asco
mpared
with
patientswho
did
notresp
ond
(22/35
;P¼
0.00
3).
(46)
BE-62(28patientswith
Barrett's
esop
hagu
sprogres
sedto
EAC
andremaining
34pa
tientswith
Barrett's
esop
hagu
swere
nonp
rogres
sors)
Methy
latio
n-sp
ecificPCR
Three-tie
redstratifi
catio
nmod
elwas
dev
elop
edus
ingmethy
latio
nindex
(p16
,HPP1,
andRUNX3),B
arrett's
esop
hagu
sleng
than
dpatho
logy
.Com
bine
dmod
elbas
edon
2-(ROC:0
.838
6)an
d4-ye
ar(ROC:0
.791
0)predictionwas
able
toca
tego
rizepatientswith
Barrett'ses
opha
gusinto
low-risk,
interm
ediate-risk,
andhigh
-riskgrou
psforEAC
dev
elop
men
t.
(44)
(Con
tinue
don
thefollo
wingpag
e)
Shah et al.
Cancer Epidemiol Biomarkers Prev; 22(7) July 2013 Cancer
Epidemiology, Biomarkers & Prevention1190
on June 7, 2021. © 2013 American Association for Cancer
Research. cebp.aacrjournals.org Downloaded from
Published OnlineFirst April 10, 2013; DOI:
10.1158/1055-9965.EPI-12-1415
http://cebp.aacrjournals.org/
-
Tab
le2.
Sum
maryof
hypermethy
latedge
nesduringBE/EAC
dev
elop
men
t(Con
t'd)
Num
ber
(%)ofsa
mplessh
owinghy
permethy
lationorstud
yfind
ings
Gen
eLo
cation
Func
tion
Metho
dNorm
alBE
LGD
HGD
EAC
Ref.
BE-195
(145
patientswith
Barrett's
esop
hagu
sprog
ressed
toEAC
andremaining
50patientswith
Barrett's
esop
hagu
swere
nonp
rogres
sors)
Methy
latio
n-sp
ecificPCR
HPP1(P
¼0.00
25),p16
(P¼
0.00
66),an
dRUNX3(P
¼0.00
02)w
eresign
ifica
ntly
hypermethy
latedin
progres
sors
asco
mpared
with
nonp
rogres
sors.In
combination,
pan
elof
8methy
latio
nmarke
rs(p16
,HPP1,
RUNX3,
CDH13
,TA
C1,
NELL
1,AKAP12
,and
SST)
show
edse
nsitivitie
sof
0.44
3an
d0.62
9at
spec
ificity
of0.9an
d0.8forEAC
progres
sion
inpatientswith
Barrett's
esop
hagu
sus
ingco
mbined
mod
eldes
igne
don
thebas
isof
2an
d4ye
arsof
follo
w-up.
(43)
EAC-41(adjace
ntno
rmal
samples
asco
ntrol)
Methy
latio
n-sp
ecificPCR
Patientsha
ving
morethan
50%
oftheirge
nesmethy
lated(APC,E
-cad
herin
,MGMT,
ER,p
16,D
AP-kinase,
andTIMP3)
show
edsign
ifica
ntly
poo
r2-ye
arsu
rvival(P
¼0.04
)and
2-ye
arrelapse
-freesu
rvival(P
¼0.03
)asco
mpared
with
thepa
tientsha
ving
less
than
50%
methy
latio
n.
(45)
BE-18,
EAC-38(m
ultip
lebiopsies
weretake
nan
dclas
sified
into
norm
al,B
arrett's
esop
hagu
s,HGD,a
ndEAC)
Bisulfite-m
odified
DNAwith
PCR
Themethy
latio
nfreq
uenc
iesof
9ge
nes(APC,C
DKN2A
,ID4,
MGMT ,
RBP1,
RUNX3,
SFR
P1,
TIMP3,
andTM
EFF
2)foun
dto
be95
%,5
9%,7
6%,5
7%,7
0%,
73%
,95%
,74%
,and
83%
,res
pec
tively,inEACsa
mples,whe
reas
95%
,28%
,78
%,4
8%,5
8%,4
8%,9
3%,8
8%,a
nd75
%,res
pec
tively,
inBarrett's
esop
hagu
ssa
mples,
which
was
sign
ifica
ntly
high
eras
compared
with
norm
alsq
uamou
sep
ithelium.T
hemethy
latio
nfreq
uenc
yforC
DKN2A
andRUNX3was
sign
ifica
ntly
high
erforEAC
asco
mpared
with
Barrett's
esop
hagu
sbiopsy
samples
.
(177
)
Normal-30,
BE-29,
HGD-8,E
AC-29
Illum
inaGolden
Gatemethy
latio
nbea
darray
Ove
rallmed
ianmethy
latio
nat
thetotal706
numbersof
mos
tinformativeCpG
sites
grad
ually
increa
sedfrom
norm
al-B
E-H
GD/EAC
(P<0.00
1).T
heau
thors
differen
tiatedbetwee
nEACvs.normal,H
GDvs.normal,B
arrett'ses
opha
gusvs.
norm
al,E
ACvs
.Barrett's
esop
hagu
s,an
dHGDvs
.Barrett's
esop
hagu
sbas
edon
422,
225,
195,
17,a
nd3nu
mbersof
CpG
sites,which
issh
owingdifferen
tial
methy
latio
nbetwee
nresp
ectiv
egrou
ps.
(178
)
Iden
tifica
tionpha
se(BE-22,
EAC-
24);retros
pec
tiveva
lidation
pha
se(BE-60,
LGD/H
GD-36,
EAC-90);p
rosp
ectiv
eva
lidation
pha
se(98pa
tientsun
der
surveillanc
e).
Iden
tifica
tionpha
se:IlluminaInfinium
assa
y;retros
pec
tive/prosp
ectiv
eva
lidationpha
se:
pyros
eque
ncing
Onthebas
isof
initial
iden
tifica
tionpha
se,7
gene
s(SLC
22A18
,ATP
2B4,
PIGR,
GJA
12,R
IN2,
RGN,a
ndTC
EAL7
)sho
wingmos
tprominen
tmethy
latio
nch
ange
swerese
lected
forva
lidation.
Com
binationof
4ge
nes(ROC
0.98
8)SLC
22A18
,PIG
R,G
JA12
,and
RIN2sh
owed
sens
itivityof
94%
andsp
ecificityof
97%.T
hispa
nelo
f4ge
nessh
owingdifferen
tialm
ethy
latio
n,stratifi
edpatients
into
low-,interm
ediate-,an
dhigh
-riskgrou
psforEAC
dev
elop
men
tin
prosp
ectiv
eva
lidation.
(179
)
Non
dys
plasticBarrett'ses
opha
gus
(not
progres
sedto
EAC)-16
,Barrett's
esop
hagu
smuc
osa
from
patientsprogres
sedto
EAC-12
Methy
latio
n-se
nsitive
sing
le-stran
dco
nformation
analysis
andmethy
latio
n-se
nsitive
dot
blot
assa
yBarrett'ses
opha
gussa
mplesco
llected
from
patientswho
prog
ressed
toEACin12
mon
thstim
epe
riodsh
owed
100%
,91%
,and
92%
hypermethy
latio
nof
APC,
TIMP-3,a
ndTE
RT,
resp
ectiv
ely,
asco
mpared
with
36%
,23%
,and
17%
inBarrett's
esop
hagu
smuc
osaco
llected
from
patientswho
did
notprogres
sto
EAC.
(180
)
Methy
lationmarke
rpan
el
Sam
ple
size
Metho
dFind
ings
Ref.
Biomarkers for Esophageal Adenocarcinoma
www.aacrjournals.org Cancer Epidemiol Biomarkers Prev; 22(7)
July 2013 1191
on June 7, 2021. © 2013 American Association for Cancer
Research. cebp.aacrjournals.org Downloaded from
Published OnlineFirst April 10, 2013; DOI:
10.1158/1055-9965.EPI-12-1415
http://cebp.aacrjournals.org/
-
Tab
le3.
Sum
maryof
gene
expressionprofilingstud
iesforBE/EAC
Sam
ple
size
Array
des
criptio
nOutco
me
Find
ings
Externa
lvalidation
Ref.
BE-21(pairedno
rmal
esop
hage
alan
dga
stric
samplesas
control)
Seriala
nalysisof
gene
express
ion,
PCRan
dim
mun
oblotting
Disea
seprog
ression
Ofno
te,5
34tags
weresign
ifica
ntly
differen
tially
expressed
betwee
nno
rmales
opha
gealsq
uamou
sep
ithelium
andBarrett's
esop
hagu
s.Th
emos
tup
regu
latedge
nesin
Barrett's
esop
hagu
sas
compared
with
norm
alep
ithelium
wereiden
tified
tobetrefoilfac
tors,a
nnex
inA10
andga
lectin-4
with
each
differen
ttypeof
tissu
esh
owed
anun
ique
cytoke
ratin
expres
sion
.
No
(181
)
Barrett's
esop
hagu
san
dHGD-11
(match
edbiopsy
samples)
cDNAmicroarray
Disea
seprog
ression
Using
2.5-fold
cutoff,iden
tified
131up
regu
latedan
d16
dow
nreg
ulated
gene
sinHGD.Twen
ty-fou
rof28
mos
tsign
ifica
ntly
differen
tge
nessh
owed
similar
chan
gesduringva
lidation.
Rea
l-tim
ePCR
(182
)
EAC-91
Oligo-microarray
Disea
seprog
ression
A4-ge
nepan
elco
nsists
ofdeo
xycy
tidinekina
se,3
0 -pho
spho
aden
osine50-pho
spho
sulfa
tesy
ntha
se2,
sirtuin-2,
andtripartitemotif-co
ntaining
44predicted
5-ye
arsu
rvival.
Immun
ohistoch
emistry
(183
)
Twen
ty-three
pairedBarrett's
esop
hagu
san
dno
rmal
epith
elium
samples
Tran
scrip
tiona
lprofiling
andproteo
mics
Disea
seprog
ression
Iden
tified
2,82
2ge
nesto
bedifferen
tially
expressed
betwee
nBarrett's
esop
hagu
san
dno
rmal
epith
elium.S
ignifica
ntly
overex
press
edge
nes
duringBarrett's
esop
hagu
sbe
long
edto
cytokine
san
dgrow
thfactors,
cons
titue
ntsof
extrac
ellular
matrix
,bas
emen
tmem
brane
andtig
htjunc
tions
,proteinsinvo
lved
inprostag
land
inan
dpho
spho
inos
itolm
etab
olism,n
itric
oxide
produc
tionan
dbioen
erge
tics.
While
gene
sen
codingHSPan
dva
rious
kina
seswere
dow
nreg
ulated
.
No
(184
)
Lymphno
demetas
tatic
(n¼
55)
andno
nmetas
tatic
(n¼
22)E
AC
samples
Oligo-microarray
Disea
seprog
ression
Lymphno
de–
pos
itive
samplessh
owed
sign
ifica
ntdow
nreg
ulationof
arginino
succ
inatesynthe
tase
asco
mpared
with
lymphno
deno
nmetas
tatic
samples(P
¼0.04
8).
No
(185
)
EAC-6
andga
stric
cardiaca
ncer-8
aCGH
Disea
seprog
ression
Iden
tified
HGF(45%
)and
BCAS1(27%
)tobemos
tfreq
uently
overex
pressed
gene
sresp
ectiv
elyat
7q21
and20
q13
locu
s.
No
(186
)
Eleve
nmatch
edsa
mplese
ts(hea
lthy-BE-EAC
match
ed-6,
norm
al-B
Ematch
ed-4
and
norm
al-EAC
match
ed-1)
SNPmicroarray
Disea
seprog
ression
60%
ofBarrett's
esop
hagu
san
d57
%of
EAC
samplesco
ntaine
dat
leas
ton
eof
thege
nomic
alteratio
nsin
theform
ofdeletions
,dup
lications
,am
plifica
tions
,cop
ynu
mber
chan
ges,
andne
utral
LOH.
No
(187
)
(Con
tinue
don
thefollo
wingpag
e)
Shah et al.
Cancer Epidemiol Biomarkers Prev; 22(7) July 2013 Cancer
Epidemiology, Biomarkers & Prevention1192
on June 7, 2021. © 2013 American Association for Cancer
Research. cebp.aacrjournals.org Downloaded from
Published OnlineFirst April 10, 2013; DOI:
10.1158/1055-9965.EPI-12-1415
http://cebp.aacrjournals.org/
-
Tab
le3.
Sum
maryof
gene
expressionprofilingstud
iesforBE/EAC
(Con
t'd)
Sam
ple
size
Array
des
cription
Outco
me
Find
ings
Externa
lvalidation
Ref.
Normal-39,
BE-25,
EAC-38,
and
ESCC-26
cDNAmicroarray
Disea
seprogres
sion
Clusteringsh
owed
these
parationof
samples
into
4distinct
grou
ps.
Ofno
te,2
,158
clon
eswere
differen
tially
expressed
betwee
nno
rmal
and
Barrett's
esop
hagu
ssa
mples,
whe
reas
1,30
6be
twee
nBarrett's
esop
hagu
san
dEAC.B
E/EAC
samplessh
owed
differen
tiale
xpressionof
hydrolase
s,lyso
zyme,
fuco
sidas
e,tran
scrip
tion
factors,
muc
ins,
andthetrefoilfac
tors.
No
(188
)
BE-20,
LGD-19,
HGD-20an
dEAC-42
SNPmicroarray
Disea
seprogres
sion
Increa
sing
numbersof
SNPsan
dloss
ofch
romos
omes
with
disea
seprogres
sion
.Chrom
osom
aldisruptio
nwas
iden
tified
intheFH
IT,
WWOX,R
UNX1,
KIF26
B,M
GC48
628,
PDE4D
,C20
orf133
,GMDS,D
MD,a
ndPARK2ge
nesin
EAC.
No
(189
)
EAC-75sp
ecim
ensfrom
64pa
tients,ad
jace
ntpairedno
rmal
tissu
efrom
patientswith
EAC-
28
DNAmicroarray
Disea
seprogres
sion
Iden
tified
AKR1B
10,C
D93
,CSPG2,
DKK3,
LUM,
MMP1,
SOX21
,SPP1,
SPARC,a
ndTW
IST1
gene
sas
biomarke
rbas
edon
tran
scrip
tomicsdata.
Qua
ntita
tivereal-tim
ePCRiden
tified
SPARC
and
SPP1ge
nesto
beas
sociated
with
EAC
patient
survival
(P<0.02
4).
Rea
l-tim
ePCR
(190
)
EAC-8,g
astric
cardia
canc
er-3
aCGH
andcD
NA
microarray
Disea
seprogres
sion
Tran
scrip
tomicsdataiden
tified
11ge
nesto
be
differen
tially
expressed
(ELF
3,SLC
45A3,CLD
N12
,CDK6,
SMURF1
,ARPC1B
,ZKSCAN1,
MCM7,
COPS6,
FDFT
1 ,an
dCTS
B).IHCan
alys
isreve
aled
sign
ifica
ntov
erex
pressionof
CDK6ace
ll-cy
cle
regu
latorin
tumor
samples.
No
(191
)
BE-20
aCGH
arrays
andhigh
den
sity
SNP
geno
typing
Disea
seprogres
sion
Cop
ynu
mber
loss
esweredetec
tedat
FRA3B
(81%
),FR
A9A
/C(71.4%
),FR
A5E
(52.4%
),an
dFR
A4D
(52.4%
)site
sin
early
Barrett's
esop
hagu
s.Validationstud
yco
nfirm
edloss
ofFR
A3B
and
FRA16
Din
early
Barrett's
esop
hagu
ssa
mples.
Rea
l-tim
ePCRan
dpyros
eque
ncing
(192
)
BE-11,
gastroes
opha
geal
junc
tion
(GEJ)
aden
ocarcino
ma-11
aCGH
with
awho
lech
romos
ome8q
contig
array
Disea
seprogres
sion
Ove
rexp
ress
ionof
MYC
andEXT1
,while
downreg
ulationof
MTS
S1,
FAM84
B,a
ndC8o
rf17
issign
ifica
ntly
asso
ciated
with
GEJ
aden
ocarcino
ma.
(193
)
BE-14,
EAC-5,E
SCC-3
cDNAmicroarray
Disea
seprogres
sion
Iden
tified
160ge
nesthat
candifferen
tiate
betwee
nBarrett's
esop
hagu
san
des
opha
geal
canc
er.
No
(194
)
Twen
ty-fou
rpa
iredsa
mplesof
norm
al,B
arrett's
esop
hagu
s,an
dEAC
phen
otyp
e
cDNAmicroarray
Disea
seprogres
sion
Ofno
te,2
14differen
tially
regu
latedge
nesco
uld
differen
tiate
betwee
nno
rmal,B
arrett'ses
opha
gus,
andEACphe
notype.
Gen
esinvo
lved
inep
idermal
No
(195
)
(Con
tinue
don
thefollo
wingpag
e)
Biomarkers for Esophageal Adenocarcinoma
www.aacrjournals.org Cancer Epidemiol Biomarkers Prev; 22(7)
July 2013 1193
on June 7, 2021. © 2013 American Association for Cancer
Research. cebp.aacrjournals.org Downloaded from
Published OnlineFirst April 10, 2013; DOI:
10.1158/1055-9965.EPI-12-1415
http://cebp.aacrjournals.org/
-
Tab
le3.
Sum
maryof
gene
expressionprofilingstud
iesforBE/EAC
(Con
t'd)
Sam
ple
size
Array
des
criptio
nOutco
me
Find
ings
Externa
lvalidation
Ref.
differen
tiatio
nareun
derex
pressed
inEAC
asco
mpared
with
Barrett's
esop
hagu
s.Exp
ress
ion
ratio
ofGATA
6to
SPRR3ca
ndifferen
tiate
betwee
n3ph
enotyp
esstud
ied.
Poo
ledbiosp
ysa
mplesfrom
Barrett's
esop
hagu
s,es
opha
geal
squa
mou
s,ga
stric
,an
dduo
den
um
Oligo-microarray
Disea
seprog
ression
Differen
tiate
differen
ttis
sueclus
ters
bas
edon
gene
expressionprofile.Iden
tified
38ge
nesthat
are
upregu
latedin
Barrett's
esop
hagu
stis
sueclus
ter,
which
belong
toce
llcy
cle(P1c
dc4
7,PCM-1),ce
llmigratio
n(urokina
se-typ
eplasm
inog
enrece
ptor,
LUCA-1/H
YAL1
),grow
thregu
latio
n(TGF-b
superfamily
protein,a
mphiregu
lin,C
yr61
),stress
resp
onse
s(calcy
clin,A
TF3,
TR3orpha
nrece
ptor),
epith
elialc
ells
urface
antig
ens(epsilon-BP,E
SA,
integrin
b4,m
esothe
linCAK-1
antig
enprecu
rsor),
and4muc
ins.
No
(196
)
Normal-24,
BE-18,
EAC-9
cDNAmicroarray
Disea
seprog
ression
Iden
tified
457,
295,
and36
differen
tially
expres
sed
gene
s,resp
ectiv
ely,betwee
nno
rmal-EAC,normal-
Barrett's
esop
hagu
s,an
dBE–EAC
grou
ps.
No
(197
)
89-EAC
cDNA-m
ediated
anne
aling,
selection,
extens
ion,
andlig
ation
assa
ywith
502kn
own
canc
er-related
gene
s
Disea
seprog
ression
Iden
tified
differen
tialg
eneex
pressionbe
twee
nea
rlystag
esof
EAC(T1an
dT2
)vs.late
(T3an
dT4
).Gen
eex
pressionprofile
reve
aled
ERBB4,
ETV
1,TN
FSF6
,MPLge
nesto
beco
mmon
betwee
nad
vanc
edtumor
stag
ean
dlymphno
demetas
tasis.
No
(198
)
Normal
esop
hage
almuc
osa-9,
esop
hagitis
-6,B
E-10,
EAC-5,
GEJad
enoc
arcino
ma-9,
stom
achsa
mples-32
(normal
muc
osa-11
,IM-9,intes
tinal-
typead
enoc
arcino
ma-7,
and
diffus
eca
rcinom
a-5)
cDNAmicroarray
Disea
seprog
ression
Onthebas
isof
theex
pressionprofile,g
enes
asso
ciated
with
thelip
idmetab
olism
andcy
tokine
nodulearefoun
dto
besign
ifica
ntly
altered
betwee
nEAC
andothe
rgrou
ps.
No
(199
)
Sev
enteen
pairedsa
mples
ofno
rmal,B
E/EAC
cDNAmicroarray
Disea
seprog
ression
Eac
htis
suetype
expresses
distin
ctse
tof
gene
s,which
candifferen
tiate
betwee
ntheirph
enotyp
es.
Barrett'ses
opha
gusan
dEACex
presses
similarset
ofstromal
gene
sthat
aredifferen
tfrom
norm
alep
ithelium.
No
(200
)
BE-19,
EAC-20(98tis
sue
spec
imen
swereco
llected
and
catego
rized
into
differen
tgrou
ps)
Onthebas
isof
previou
smicroarraystud
ies23
gene
swereva
lidated
usingreal-tim
ePCR
Disea
seprog
ression
Out
of23
gene
s,pa
nelo
f3ge
nes(BFT
,TSPAN,a
ndTP
)was
able
todiscrim
inatebetwee
nBarrett's
esop
hagu
san
dEACin
internal
valid
ationwith
0%clas
sifica
tionerror.
N.A.
(201
)
(Con
tinue
don
thefollo
wingpag
e)
Shah et al.
Cancer Epidemiol Biomarkers Prev; 22(7) July 2013 Cancer
Epidemiology, Biomarkers & Prevention1194
on June 7, 2021. © 2013 American Association for Cancer
Research. cebp.aacrjournals.org Downloaded from
Published OnlineFirst April 10, 2013; DOI:
10.1158/1055-9965.EPI-12-1415
http://cebp.aacrjournals.org/
-
Tab
le3.
Sum
maryof
gene
expressionprofilingstud
iesforBE/EAC
(Con
t'd)
Sam
ple
size
Array
des
cription
Outco
me
Find
ings
Externa
lvalidation
Ref.
Normal-30,
BE-31,
gastric
muc
osa-34
,duo
den
um-18
Biomarke
rsforBarrett's
esop
hagu
swere
iden
tified
using3
pub
licly
available
microarraydatas
ets
andva
lidated
using
real-tim
ePCRan
dim
mun
ohistoch
emistry.
Disea
seprogres
sion
Out
of14
gene
siden
tified
,dop
ade
carbox
ylas
e(DDC)
andTrefoilfac
tor3(TFF
3)wereva
lidated
tobe
upregu
latedin
Barrett's
esop
hagu
s.
N.A.
(202
)
EAC-56
Olig
onuc
leotide
microarrayan
daC
GH
Disea
seprogres
sion
Iden
tified
4ne
wge
nes(EGFR
,WT1
,NEIL2,
and
MTM
R9)to
beov
erex
pressed
in10
%to
25%
EAC.
Exp
ress
ionleve
lsof
thes
e4ge
nesdifferen
tiated
patie
ntswith
EAC
into
3grou
psna
melygo
od,
averag
e,an
dpoo
rdep
endingup
ontheirp
rogn
osis
(P<0.00
8)
Immun
ohistoch
emistry
(203
)
BE/LGD-72,
HGD-11,
EAC-15
Bac
teria
lartificial
chromos
omeaC
GH
Disea
seprogres
sion
Cop
ynu
mber
chan
gesweremoreco
mmon
and
larger
asdisea
seprogres
sto
laters
tage
s.Patients
having
copynu
mber
alteratio
nsinvo
lvingmore
than
70Mbpwereat
increa
sedris
kof
progres
sion
toEAC
(P¼
0.00
47)
No
(60)
EAC-30,
BE-6,L
GD-9,H
GD-10
Gen
ome-wideCGH
Disea
seprogres
sion
Loss
of7q
33-q35
was
foun
din
HGDas
compared
with
precu
rsor
LGD(P
¼0.01
).Lo
ssof
16q21
-q22
andga
inof
20q1
1.2-q1
3.1was
sign
ifica
ntly
differen
tbetwee
nHGDan
dEAC
(P¼
0.02
and
0.03
,res
pec
tively).
No
(56)
EAC-30,
lymphno
demetas
tasis-
8,HGD-11,
LGD-8,a
ndBE-6
from
30EAC
patient
biopsy
samples
CGH
Disea
seprogres
sion
Iden
tified
region
sun
dergo
ingco
pynu
mber
loss
and
amplifica
tionduringea
chstag
eof
tran
sitio
n.Ave
rage
number
ofch
romos
omal
imba
lanc
ese
que
ntially
increa
sedfrom
BE–LG
D–HGD–EAC–
lymphno
demetas
tasis.
No
(54)
Forty-tw
opatientsreprese
ntdifferen
tstag
esof
disea
seSNParray
Disea
seprogres
sion
SNPab
norm
alities
increa
sesfrom
2%to
morethan
30%
asthedise
aseprog
ress
from
Barrett's
esop
hagu
sto
EAC.T
otalnu
mbe
rofS
NPalteratio
nsin
tissu
esa
mples
istig
htlyco
rrelated
with
DNA
abno
rmalities
such
asan
euploidy
andLO
H.
No
(57)
EAC-27an
dmatch
edno
rmal-14
SNParray
Disea
seprogres
sion
Con
firm
edprev
ious
lydes
cribed
geno
mic
alteratio
nssu
chas
amplifica
tionon
8qan
d20
q13
ordeletion/
LOH
on3p
and9p
.Alsoiden
tified
alteratio
nsin
seve
raln
ovel
gene
san
dDNAregion
sin
EAC
samples
.
No
(58)
(Con
tinue
don
thefollo
wingpag
e)
Biomarkers for Esophageal Adenocarcinoma
www.aacrjournals.org Cancer Epidemiol Biomarkers Prev; 22(7)
July 2013 1195
on June 7, 2021. © 2013 American Association for Cancer
Research. cebp.aacrjournals.org Downloaded from
Published OnlineFirst April 10, 2013; DOI:
10.1158/1055-9965.EPI-12-1415
http://cebp.aacrjournals.org/
-
Tab
le3.
Sum
maryof
gene
expressionprofilingstud
iesforBE/EAC
(Con
t'd)
Sam
ple
size
Array
des
cription
Outco
me
Find
ings
Externa
lvalidation
Ref.
EAC-26
SNParray
Disea
seprogres
sion
Con
firm
edpreviou
slyreportedfreq
uent
chan
gesto
FHIT,C
DKN2A
,TP53
,and
MYC
gene
sin
EAC.
Iden
tified
PDE4D
andMGC48
628as
tumor-
suppress
orge
nes.
No
(59)
EAC-35
cDNAmicroarray
Res
pon
seto
chem
othe
rapy
Iden
tified
165differen
tially
expres
sedge
nesbetwee
npo
or(n
¼17
)and
good
outcom
e(n
¼18
)patient
grou
ps.
Topfunc
tiona
lpathw
aybas
edon
differen
tialg
eneex
pressionwas
iden
tified
tobe
Toll-rece
ptorsign
aling.
No
(204
)
EAC-47(lo
cally
adva
nced
tumor)
cDNAmicroarray
Res
pon
seto
chem
othe
rapy
Iden
tified
86ge
nessh
owingat
leas
t2-folddifferen
cebe
twee
nch
emothe
rapy
resp
onders(n
¼28
)and
nonres
pon
ders(n
¼19
).EphrinB3rece
ptor,w
hich
show
edhigh
estdifferen
cebetwee
nthegrou
ps,
show
edstrong
mem
brane
staining
inch
emothe
rapyresp
ondingtumorsus
ing
immun
ohistoch
emistry.
No
(205
)
Patientswith
EAC-19un
dergo
ing
chem
orad
iotherap
yOlig
o-microarray
Res
pon
seto
chem
orad
iotherap
yRed
uced
expressionof
IVL,
CRNN,N
ICE-1,S
100A
2,an
dSPPR3ge
nesco
rrelated
with
poo
rsurviva
land
nonres
pon
seto
chem
othe
rapy.
No
(206
)
19patients(EAC-16,
ESCC-2
and
aden
osqua
mou
sca
rcinom
a-1)
undergo
ingch
emorad
iotherap
y
Olig
o-microarray
Res
pon
seto
chem
orad
iotherap
yLo
wer
expressionforp
anelof
gene
sPERP,S
100A
2,an
dSPRR3was
asso
ciated
with
nonres
pon
seto
therap
y.Pathw
ayan
alysis
iden
tified
downreg
ulationof
apop
tosisin
nonres
pon
ders.
No
(207
)
EAC-174
,ESCC-36
SNPsas
sociated
with
the
chem
othe
rapy
drug
actio
npa
thway
Res
pon
seto
chem
orad
iotherap
yIden
tified
asso
ciationbetwee
nge
netic
polymorphism
san
dresp
onse
topreop
erative
chem
othe
rapy(fluo
rourac
ilan
dplatinum
compou
nds)
andradiotherap
y.
No
(208
)
NOTE
:Majority
ofstud
iesdes
cribed
inthis
table
includ
eva
lidationof
resu
ltsin
thesa
mepatient
coho
rtas
used
indisco
very
pha
se.Onlystud
iesthat
includ
edva
lidationus
ing
indep
enden
tpatient
coho
rtarede
scrib
edas
external
valid
ation.
Shah et al.
Cancer Epidemiol Biomarkers Prev; 22(7) July 2013 Cancer
Epidemiology, Biomarkers & Prevention1196
on June 7, 2021. © 2013 American Association for Cancer
Research. cebp.aacrjournals.org Downloaded from
Published OnlineFirst April 10, 2013; DOI:
10.1158/1055-9965.EPI-12-1415
http://cebp.aacrjournals.org/
-
decade, several studies conducted using advanced geno-mic
techniques such as array-comparative genomichybridization (aCGH)
and SNP arrays confirmed previ-ously reported copy number
alterations and identifiednovel genomic loci undergoing changes
during process ofmetaplasia–dysplasia–carcinoma development
(54–60). Ithas been shown that as the disease progresses from
earlyto late stages, SNP abnormalities increase from approxi-mately
2% to 30% (54, 57). The total number of SNPalterations in tissue
samples is tightly correlated withpreviously reported DNA
abnormalities such as aneu-ploidy, copy number alterations, and LOH
highlightingthe application of SNP-based genotyping to assess
geno-mic abnormalities (54–60). Thus, SNP-based genotypingprovides
an alternative way to assess genomic abnormal-ities during EAC
pathogenesis.Studies on gene expression changes in EAC have
been
propelled by recent progress in genomic technologies,each
identifying unique sets of gene expression profile,which can be
used as a biomarker panel for diseasediagnosis, prognosis, or to
predict response to therapy(Table 3).Moreover, determination of the
gene expressionchanges has been extremely helpful to
understanddetailed pathogenesis and will form basis for
developingfuture therapies. However, future validation using
inde-pendent sample cohorts will be necessary for themajorityof
these potential biomarkers.Apart from genomic abnormalities
associated with the
disease progression, inheriting genetic factors are
alsoimplicated for EAC development. Risk for BE/EAC andGERD is
increased by 2- to 4-fold when a first-degreerelative is already
affected by any of these conditions (61).Recently, a study
conducted by The Esophageal Adeno-carcinoma Genetics Consortium and
TheWellcome TrustCase Control Consortium identified link between
SNPs atthe MHC locus and chromosome 16q24.1 with risk forBarrett’s
esophagus (62). They also identified SNPs asso-ciated with body
weight measures that were present withmore than expected frequency
in Barrett’s esophagussamples supporting epidemiologic findings
about obesityas a risk factor for Barrett’s esophagus and EAC (62).
Wuand colleagues examined the relationship between pres-ence of
risk genotypes and the onset of EAC. They iden-tified 10 SNPs
associatedwith the age of EAConset. Genesassociated with 5 of 10
SNPs identified were known to beinvolved in apoptosis
(63).Recently, published cancer genome–sequencing stud-
ies have given deeper insights into the genomic abnor-malities
associated with the EAC pathogenesis. The com-parative genomic
analysis between EAC and ESCCreported by Agrawal and colleagues
(64) confirmed pre-viously verywell-described association of p53
genemuta-tions with esophageal cancer development. The authorsalso
conducted comparative genome-wide analysisbetween matched Barrett’s
esophagus and EAC patienttissue samples and concluded that the
majority of geno-mic changes occur early during EAC development, at
thestageofBarrett’s esophagus (64). Similar conclusionswere
made by next-generation sequencing of biopsy samplesobtained
from the same patient at the stage of Barrett’sesophagus and EAC
(65). The authors also identifiedARID1A as novel tumor-suppressor
gene and around15% of patientswith EAC showed loss of
ARID1Aproteinin tissue samples. In vitro studies suggested it to
beassociated with cell growth, proliferation, and invasion(65).
Very recently published high-resolution methylomeanalysis has
provided first evidence for methylationchanges at genomic regions
that encodenoncodingRNAs.The authors identified
longnoncodingRNA,AFAP1-AS1,to be severely hypomethylated in
Barrett’s esophagus andEAC tissue samples, silencing of which
significantlyreduced aggressiveness of EAC cell lines OE33 andSKGT4
(66).
Taken together, genomic abnormalities play key rolesduring each
stage of transformation from normal squa-mous epithelium to
EAC.
Cancer-Related InflammationGastric and bile acid exposure in the
esophageal epi-
thelium leads to the development of chronic inflamma-tory
conditions mainly driven by elevated levels of proin-flammatory
cytokines. Chronic inflammatory responsesinduce cell survival and
increase cell proliferation, henceplay key roles in the development
of EAC (67, 68). Expres-sions of various inflammatory molecules
such as COX-2,NF-kB, interleukin (IL)-6, IL-8, and matrix
metalloprotei-nases (MMP) have been evaluated as prognostic
biomar-kers for BE/EAC development.
Exposure to gastric/bile acid and cytokines leads toincreased
COX-2 expression (69). COX-2 is a rate-limitingenzyme that
regulates synthesis of prostaglandins fromarachidonic acid. COX-2
directly increases cell prolifera-tion and promotes tumor invasion
(69), andCOX-2–medi-ated increase in prostaglandin synthesis could
result intumor growth and angiogenesis (70). COX-2 expressionhas
been detected in disease-free esophageal tissue homo-genates using
immunoblotting (69). In comparison withGERD, patients suffering
from erosive reflux show slight-ly higher gene expressions of this
enzyme in tissue sam-ples (71). Several studies have shown
significantlyincreased COX-2 expression correlating with the
diseaseprogression from Barrett’s esophagus to dysplasia andEAC
(69, 72–75). Furthermore, expression levels of COX-2have been shown
to have a prognostic value in EAC withhigher levels associated with
poor survival and increasedchances of tumor relapse (76, 77).
Another well-studied inflammatory biomarker NF-kBis activated in
response to exposure with bile acid andelevated NF-kB expression
levels are found during Bar-rett’s esophagus, dysplasia, and
adenocarcinoma (78–80).Activated NF-kB translocates from cytoplasm
to nucleusand upregulates transcription of the genes involvedin
inflammatory processes. Moreover, nuclear NF-kBexpression has been
shown to be correlated with thepatient response to
chemoradiotherapy.All of thepatientswho showed complete response to
chemoradiotherapy
Biomarkers for Esophageal Adenocarcinoma
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July 2013 1197
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http://cebp.aacrjournals.org/
-
had elevated NF-kB levels pretreatment and showed lackof active
NF-kB posttreatment (81).
In line with NF-kB and COX-2, expression of indi-vidual or
combinations of proinflammatory cytokinesIL-1b, IL-6, IL-8, and
TNF-a is significantly increased inBarrett’s esophagus and EAC as
compared with squa-mous epithelium (82–84). IL-1b and IL-8
expressionlevels also correlate with the stage of EAC (79).
Patientswho responded to neoadjuvant chemotherapy treat-ment showed
significantly reduced expressions of IL-8 and IL-1b in
postchemotherapy esophageal tissuesections (81). IL-6 is activated
in response to reflux andthe IL-6/STAT3 antiapoptotic pathway may
underliethe development of dysplasia and tumor (85). Serum IL-6
levels were reported to provide 87% sensitivity and92% specificity
for EAC diagnosis in a recent retrospec-tive study (86). However,
the study only comparedbetween healthy and EAC groups. It would be
interest-ing to see how early it can diagnose EAC during theprocess
of metaplasia–dysplasia. Combination of cyto-kines IFN-g , IL-1a,
IL-8, IL-21, and IL-23 along withplatelet proteoglycan and
miRNA-375 expression pro-filing has been shown to build an
inflammatory riskmodel, which has clinical use to determine
prognosis forpatients with EAC (67).
MMPs are a family of proteolytic enzymes involved inthe
degradation of extracellular matrix components.MMPs play a role in
both inflammation and tumormetas-tasis. Immunohistochemical
staining forMMP-1, MMP-2,MMP-7, andMMP-9 has been reported to be
significantlyhigher in EAC as compared with healthy individuals
(87,88). Higher level of MMP-1 expression has been associ-ated with
the lymph node metastases and possibly poorpatient survival (89).
Expression ofMMP-9 is shown to bean early event during the EAC
transformation and itsexpression levels are correlated with the
progression ofthe disease (90–92). Activity of MMPs is inhibited by
afamily of proteins called tissue inhibitors of metallopro-teinases
(TIMP). Specifically, TIMP-3 gene is methylatedin EAC development
and its reduced expression is asso-ciated with stage of the tumor
and patient survival (93).On contrary, Salmela and colleagues
described elevatedTIMP-1 and TIMP-3 expression in EAC tumor
samples(88).
Although the underlying tissue inflammation is veryclosely
associated with EAC development and severalinflammation-related
biomarkers have been identified,these remain to be validated in
large-scale biomarkerstudies.
Cell Cycle–Related AbnormalitiesTo compensate for the tissue
damage induced by gas-
tric/bile acid, the underlying epithelium starts to prolif-erate
rapidly and become uncontrolled resulting in neo-plasia. To meet
the proliferation requirements, the cellshave to overcome
cell-cycle checkpoints. Cyclin D1 over-expression is one such means
by which cells overcomeG1–S checkpoint, and cyclin D1
immunohistochemical
staining has been proposed to identify patients with Bar-rett’s
esophagus with an increased risk for EAC (94). Incontrast to cyclin
D1, expression of p16 protein results incell-cycle arrest in G1
phase as it has been shown to inhibitcyclin-dependent
kinase–induced phosphorylation ofretinoblastoma protein. Early
genomic abnormalities dur-ing EAC development significantly affect
p16 proteinexpression,which can bedeterminedusing immunostain-ing
and implemented as a potential biomarker (95). Fur-ther large-scale
trials are required to confirm cell-cycleabnormalities during EAC
development to implementthem as a biomarker.
Bottom of the pyramid in Fig. 1 represents list ofbiomarkers in
the initial stages of development. Tumorsharboring overexpression
of growth factor receptors [EGFreceptor (EGFR) and HER-2] are
associated with poorpatient survival (96, 97), whereas those
overexpressingapoptosis regulator Bcl-2 protein showed prolonged
sur-vival (98). Incipient angiogenesis is a marked feature
ofBarrett’s esophagus and underlining tissue expressesangiogenesis
markers VEGF and its receptors (99). Neo-vascularization continues
as the disease progresses fromBarrett’s esophagus to EAC. Measuring
the degree ofneovascularization correlated with histopathologic
gradeof the tumor and associated with the patient survival(100).
Expression of 2 prominent cell proliferation mar-kers, PCNA and
Ki-67, has been described to be alteredduring BE–EAC development
(101).
miRNAmiRNA was first discovered in Caenorhabditis elegans
(102) and since then it has beenwidely studied in a varietyof
biologic phenomena. These short stretches of approx-imately 21
nucleotides do not code for protein but playimportant roles in gene
regulation by either suppressingprotein synthesis or causing mRNA
cleavage. UnlikesiRNA, miRNA can target multiple genes on remote
lociand therefore control diverse group of proteins. Severalkey
properties of carcinogenesis have been shown to beregulated via
miRNA, for example, angiogenesis andmetastasis (103).
With increased biologic understanding ofmiRNAs andtheir role in
cancer, they have been proposed in severaldifferent clinical
applications including cancer diagnosisand tumor prognosis, tumor
classification, and also as atherapeutic target for disease
intervention. Differentialtissue miRNA expression has been observed
in severaldifferent malignancies and these changes can be used
fordiagnosis and classification of the tumors (103). miRNAbioarrays
were first used to show differential miRNAexpression in healthy,
Barrett’s esophagus, and EACtissue samples (104). Since then, a
number of differentstudies have identified miRNA changes associated
withthe development of the BE–EAC. Table 4 summarizesprimary
findings of miRNA expression profiling studiesalong with
statistical significance and fold-change values.Biologic
significance for some of the miRNA-relatedchanges is discussed
later.
Shah et al.
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Smith and colleagues identified reduced expression ofmiR-200 and
miR-141 in Barrett’s esophagus and EACtissue samples. They
conducted bioinformatics analysisand correlated these miRNA
expression changes withcellular processes such as cell cycle, cell
proliferation,apoptosis, and cell migration (105). miR-196a, which
isdescribedas amarker of progression fromBarrett’s esoph-agus to
EAC, can increase cell proliferation and anchor-age-independent
growth and inhibit apoptosis in EACcell lines in vitro (106). The
downstream targets for miR-196a are verified to be Annexin A1, S100
calcium-bindingprotein A9, small proline-rich protein 2C, and
Keratin 5,which showed reduced expression in EAC patient
tissuesamples as compared with normal epithelium (106, 107).Several
studies described in Table 4 report overexpressionof miR-192 during
EAC carcinogenesis. miR-192 has beenreported tobe a target of p53
andhas been able to suppresscancer progression in osteosarcoma and
colon cancer celllines throughp21 accumulation and cell-cycle
arrest (108).As shown in Table 4,miR-21 is overexpressed during
BE/EAC and it can function as an oncogene as shown intumors of
breast, brain, lung, prostate, pancreas, colon,liver, and chronic
lymphocytic leukemia. It negativelyregulates tumor- and
metastasis-suppressor genes PTEN,TPM1, PDCD4, and Sprouty2
(109–112). miR-194 expres-sion is regulated by hepatocyte nuclear
factor (HNF)-1atranscription factor, which is induced during
BE/EACand may lead to upregulation of miR-194 (109).
Higherexpression of miR-194 is also observed in
metastaticpancreatic cell lines (113). Among miRNAs found to
bedownregulated during EAC development, let-7 family ofmiRNAs is
tumor-suppressive and negatively regulatesRas oncogene. Fassan and
colleagues confirmed upregu-lation of HMGA2, which is one of the
target of let-7miRNA, using immunohistochemistry in tissue
samples(110, 112, 114). Further studies in the regards of
miRNAandmiRNA target geneswill improve the biologic under-standing
of EAC pathogenesis and may also providenovel molecular targets for
disease intervention.Notably, miRNAs are found to be stable in
serum
encapsulated in microvesicles and can be accessed easily(115).
In fact, circulating miRNA profiling has showndistinct expression
patterns in a number of cancers, otherthan EAC (116). This opens up
new avenues for circulat-ing miRNA changes as a potential biomarker
for EAC.
GlycoproteinsProtein glycosylation is a common
posttranslational
modification with almost half of the proteins
synthesizedundergoing 1 of the 2 major types either N-linked or
O-linked glycan modifications. The biosynthetic process
ofglycosylation is regulated by the expression and localiza-tion of
glycosyltransferases/glycosidases and the avail-ability of
substrate glycans (117).Aberrant glycosylation changes have
previously been
reported in several different cancers namelybreast
cancer,prostate cancer, melanoma, pancreatic cancer, ovariancancer,
etc. (118, 119). These changes include truncated
forms of O-glycans, increased degree of branching in N-glycans,
and elevated sialylation, sulfation, and fucosyla-tion with a range
of other possible variations (119). Thedifferential glycosylation
can alter protein interactions,stability, trafficking,
immunogenicity, and function (118).Tumor-specific glycosylation
changes are activelyinvolved in neoplastic progression, namely
metastasis,as glycoproteins are found abundantly on cell
surfacesand extracellularmatrices and therefore play a vital role
incellular interactions.
Lectins are a family of glycan-binding proteins exten-sively
used in glycobiology due to preferential binding ofeach lectin to
recognize specific glycan structures (119,120). The first effort to
identify differential glycosylationin the progression to Barrett’s
esophagus and EAC wasmade in 1987 by Shimamoto and colleagues using
differ-ential binding pattern to 5 lectins in tissue
specimens(121). The glycoconjugate expression profile in
Barrett’sesophagus was found to be significantly different
fromnormal esophageal epithelium. Interestingly, glycoconju-gate
expression between Barrett’s esophagus and normalduodenum was quite
similar. There were minimal glyco-conjugate expression changes
between Barrett’s esopha-gus and LGD. However, EAC tissue samples
showedsignificantly different lectin-binding pattern than BE/LGD
(121). Using rabbit esophageal epithelium, Poor-khalkali and
colleagues showeddifferential lectin bindingin response to
acid/pepsin exposure suggesting acidexposure can induce cell
surface glycosylation changes(122). In 2008, Neumann and colleagues
used 4 differentlectins to identify pathologic mucosal changes
(123). Theyobserved 2 distinct lectin-binding patterns. Onewas
asso-ciated with the GERD, whereas the other pattern
wascharacteristic for Barrett’s esophagus mucosa. Specifical-ly,UEA
(Ulex europaeus) lectin bindingwasupregulated inBarrett’s esophagus
tissue sections, which suggests pos-sible increase in fucosylation
during the disease progress(123). A recently published study has
concluded thatdysplasia can alter glycan expression and lectin
bindingto the tissue samples. Fluorescently labeled WGA (wheatgerm
agglutinin) lectin-binding intensity was found to beinversely
related to the degree of dysplasia (124). Further-more, the authors
used fluorescent-capable endoscope exvivo in the study and followed
all the protocols in amanner that exactly mimics a clinical study
in vivo. Fol-lowed by topical fluorescein-labeled WGA spray,
theauthors measured fluorescence in the tissue samples.Measurement
of lectin fluorescence was a more sensitiveapproach to identify
dysplastic lesions as compared withwhite light endoscopic
technique. Their data show clinicaluse of such a lectin-based
endoscopic technique if devel-oped further (124). In a phase III
biomarker clinical trialstudy, Bird-Liberman and colleagues
combined 3 differ-ent abnormalities to predict EAC progression in
patientswith Barrett’s esophagus. Along with using conventionalLGD
and DNA content abnormalities they used AOL(Aspergillus oryzae)
lectin binding to the tissue samples,which detects presence of a1-6
fucose on the cell surface
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Table 4. Summary of literature describing miRNA expression
changes in BE/EAC
Sample size Upregulated in BE/EAC Downregulated in BE/EAC
Ref.
71 (BE-12, Barrett'sesophagus withoutdysplasia-20,
LGD-27,EAC/HGD-12)
miR-192 (P < 0.00001), miR-196a (P < 0.05):upregulated in
Barrett's esophagus ascompared with healthy
tissue.miR-196aexpression is correlated with progressionfrom
IM-LGD-HGD-EAC (P < 0.005).
miR203 (P < 0.00001): downregulation inBarrett's esophagus as
compared withhealthy tissue.
(209)
22 (Barrett's esophaguswithout dysplasia-11,Barrett's esophagus
withdysplasia-11)
miR-15b (3.3-fold; P < 0.05), miR-203 (5.7-fold; P <
0.05): upregulated in dysplasia ascompared with nondysplastic
Barrett'sesophagus.
miR-486-5p (4.8-fold; P < 0.05), miR-let-7a(3.3-fold; P <
0.05): downregulated indysplasia as compared with
nondysplasticBarrett's esophagus.
(110)
100 (EAC-100, adjacentnormal tissue as control)
miR-21 (�3-fold; P < 0.05), miR-223 (�2-fold;P < 0.05),
miR-192 (�3.5-fold; P < 0.05),and miR-194 (�3.5-fold; P <
0.05):upregulated in EAC as compared withadjacent normal
tissue.
miR-203 (�3-fold; P < 0.05): downregulatedin EAC as compared
with adjacent normaltissue.
(111)
25 (Healthy-9, BE-5,HGD-1, EAC-10)
miR-192 (1.7-fold; FDR < 1 e�07), miR-194(2-fold; FDR <
1e�07), miR-21 (3.7-fold;FDR ¼ 0.0003), miR-200c (1.9-fold; FDR
¼0.0015), miR-93 (1.3-fold; FDR ¼ 0.0108):upregulated in EAC as
compared withBarrett's esophagus.
miR-27b (1.43-fold; FDR ¼ 0.0003), miR-342(1.25-fold; FDR ¼
0.0015), miR-125b (2-fold; FDR ¼ 0.0108), miR-100 (1.25-fold;FDR ¼
0.011): downregulated in EAC ascompared with Barrett's
esophagus.
(104)
75 (Healthy-15, BE-15,LGD-15, HGD-15,EAC-15)
miR-215 (62.8-fold; P < 1e�07), miR-192(6.34-fold; P <
1e�07): upregulated inBarrett's esophagus in comparison withnormal
tissue and remained at similar levelswith disease progress.
miR-205 (10-fold; P ¼ 1.39e�0.5), let-7c(2.04-fold; P ¼
3.11e�05), miR-203 (6.67-fold; P ¼ 3.2e�0.5): downregulated
inBarrett's esophagus in comparison withnormal tissue and remained
at similar levelsas disease progresses.
(114)
91 (LGD-31, HGD-29, EAC-31, In all cases adjacentnormal tissue
used as acontrol)
miR-200a (13.5-fold; P ¼ 0.02), miR-513(1.58-fold; P ¼ 0.03),
miR-125b (9.2-fold; P¼ 0.04), miR-101 (1.83-fold; P¼ 0.04), miR-197
(1.61-fold; P ¼ 0.04): upregulated inLGD to HGD transition.
miR-23b (1.45-fold; P ¼ 0.007), miR-20b(1.56-fold; P¼ 0.01),
miR-181b (2.22-fold;P¼ 0.03), miR-203 (1.49-fold; P¼ 0.03),
miR-193b (2.70-fold; P ¼ 0.04), miR-636 (4.17-fold; P ¼ 0.04):
downregulated in LGD toHGD transition. let-7a (1.75-fold; P ¼
0.01),let-7b (1.59-fold; P ¼ 0.009), let-7c (1.69-fold; P ¼ 0.03),
let-7f (1.69-fold; P ¼ 0.03),miR-345 (2-fold; P ¼ 0.02), miR-494
(1.72-fold; P ¼ 0.03), miR-193a (2.27-fold; P ¼0.05): downregulated
in HGD-EACdevelopment process.
(112)
48 (BE-19, EAC-29) miR-21 (�2.8-fold; P < 0.05),
miR-143(�11.3-fold;P
-
(125). Thus, monitoring tissue glycan changes can becombined
with existing biomarkers to improve the pre-dictive power of the
currently used biomarkers.A potential mechanism responsible for
these changes is
considered to be bile acid exposure-induced gene expres-sion and
secretory pathway changes in esophageal epi-thelium. Using
carbohydrate-specific lectins that detectN- and O-linked
glycosylation and core fucosylation,Byrne and colleagues have shown
differential lectin bind-ing to the cell surface and differential
intracellular local-ization when normal squamous and Barrett’s
metaplasticcell lines were treated with deoxycholic acid (126).
Nan-carrowand colleagues profiledwhole-genome expressionin normal
squamous esophageal epithelium, Barrett’sesophagus, and EAC and
concluded that Barrett’s esoph-
agus is a tissue with enhanced glycoprotein synthesismachinery
to provide strong mucosal defense againstacid exposure (127).
Outlook—Circulating BiomarkersLast 3 decades showed continuously
increased EAC
incidences and similar trend is expected in future becauseof
rising incidences of obesity and GERD in the popula-tion.Current
endoscopic screeningprogrammightbenefitthe highest risk population
to monitor disease progres-sion. Monitoring dysplasia in the tissue
samples has notprovided fruitful outcome for early diagnosis;
however,inclusion of the genomic and cell-cycle biomarkers hasshown
definite improvement in the predictive powerover currently used
histologic technique. Any biomarker
Table 4. Summary of literature describing miRNA expression
changes in BE/EAC (Cont'd)
Sample size Upregulated in BE/EAC Downregulated in BE/EAC
Ref.
11 (EAC-11, differentlesions were collectedfrom these patients
andclassified into Barrett'sesophagus, LGD, HGD,and EAC)
miR-196a is overexpressed in early EAC(151-fold) > HGD
(62.2-fold; P ¼ 0.00002) >LGD (31.1-fold; P ¼ 0.0005) >
Barrett'sesophagus (28.9-fold; P ¼ 0.00001). Foldchanges are
calculated as compared withnormal epithelium.
— (107)
45 (patients with EACundergoing surgery)
miR-143 (P ¼ 0.0148), miR-199a_3p (P ¼0.0009), miR-199a_5p (P ¼
0.0129), miR-100 (P ¼ 0.0022) and miR-145 (P ¼
0.1176)expression