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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
www.aacrjournals.org 1185
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/
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
on June 7, 2021. © 2013 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from
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http://cebp.aacrjournals.org/
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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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
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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
www.aacrjournals.org Cancer Epidemiol Biomarkers Prev; 22(7) July 2013 1189
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
(%)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
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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
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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
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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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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.
Cancer Epidemiol Biomarkers Prev; 22(7) July 2013 Cancer Epidemiology, Biomarkers & Prevention1198
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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