Jou nal o
Pe sonalized
Medicine
A icle
The mal Liquid Biopsy (TLB) Focused on Benign and
P emalignan Panc ea ic Cys Diagnosis
Sonia He moso-Du án1,2,† , Guille mo Ga cía-Rayado 1,3,4,†, Lau a Ceballos-Lai a 1,2 , Ca los Sos es 1,3,4,
Sonia Vega 2, Judi h Millas e 1,3, Osca Sánchez-G acia 5, Jo ge L. Ojeda 6,Ángel Lanas 1,3,4,7 ,
Ad ián Velázquez-Campoy 1,2,4,8,9,* and Olga Abian 1,2,4,8,10,*


Ci a ion: He moso-Du án, S.;
Ga cía-Rayado, G.; Ceballos-Lai a, L.;
Sos es, C.; Vega, S.; Millas e, J.;
Sánchez-G acia, O.; Ojeda, J.L.; Lanas,
Á.; Velázquez-Campoy, A.; e al.
The mal Liquid Biopsy (TLB)
Focused on Benign and P emalignan
Panc ea ic Cys Diagnosis. J. Pe s.
Med. 2021,11, 25. h ps://doi.o g/
10.3390/jpm11010025
Recei ed: 9 Decembe 2020
Accep ed: 29 Decembe 2020
Published: 31 Decembe 2020
Publishe ’s No e: MDPI s ays neu-
al wi h ega d o ju isdic ional clai-
ms in published maps and ins i u io-
nal a ilia ions.
Copy igh : © 2020 by he au ho s. Li-
censee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and con-
di ions o he C ea i e Commons A -
ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
1Ins i u o de In es igación Sani a ia A agón (IIS A agón), 50009 Za agoza, Spain;
[email p o ec ed] (S.H.-D.); [email p o ec ed] (G.G.-R.);
[email p o ec ed] (L.C.-L.); [email p o ec ed] (C.S.); millas [email p o ec ed] (J.M.);
alanas@uniza .es (Á.L.)
2Join Uni s IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Ins i u e o Biocompu a ion and Physics o Complex
Sys ems (BIFI), Uni e sidad de Za agoza, 50018 Za agoza, Spain; [email p o ec ed]
3Se icio de Diges i o, Hospi al Clínico Uni e si a io Lozano Blesa (HCULB), 50009 Za agoza, Spain
4Cen o de In es igación Biomédica en Red en el Á ea Temá ica de En e medades Hepá icas y
Diges i as (CIBERehd), 28029 Mad id, Spain
5SOTER BioAnaly ics, En ique Val, 50011 Za agoza, Spain; osca [email p o ec ed]
6Depa men o S a is ical Me hods, Uni e sidad de Za agoza, 50009 Za agoza, Spain; jojeda@uniza .es
7Depa men o Medicine, Uni e si y o Za agoza, 50009 Za agoza, Spain
8Fundación ARAID, Gobie no de A agón, 50009 Za agoza, Spain
9Depa amen o de Bioquímica y Biología Molecula y Celula , Uni e sidad de Za agoza,
50009 Za agoza, Spain
10 Ins i u o A agonés de Ciencias de la Salud (IACS), 50009 Za agoza, Spain
*Co espondence: ad ian c@uniza .es (A.V.-C.); oabi a@uniza .es (O.A.);
Tel.: +34-976-762996 (A.V.-C.); +34-876-555417 (O.A.)
† S.H.-D. and G.G.-R. con ibu ed equally o his wo k and a e bo h i s au ho s o he manusc ip .
Abs ac :
Backg ound: Cu en e o s in he iden i ica ion o new bioma ke s a e di ec ed owa ds
an accu a e di e en ia ion be ween benign and p emalignan cys s. The mal Liquid Biopsy (TLB)
has been p e iously applied o in lamma o y and umo diseases and could o e an in e es ing poin
o iew in his ype o pa hology. Me hods: In his wo k, wen y pa ien s (12 males and 8 emales,
a e age ages 62) diagnosed wi h a panc ea ic cys benign (10) and p emalignan (10) cys lesions we e
ec ui ed, and biological samples we e ob ained du ing he endoscopic ul asonog aphy p ocedu e.
Resul s: P o eomic con en o cys liquid samples was s udied and se e al common p o eins in he
di e en g oups we e iden i ied. TLB cys liquid p o iles e lec ed p o ein con en . Also, TLB se um
sco e was able o disc imina e be ween heal hy and cys s pa ien s (71% sensi i i y and 98% speci ici y)
and be ween benign and p emalignan cys s (75% sensi i i y and 67% speci ici y). Conclusions: TLB
analysis o plasma ic se um sample, a quick, simple and non-in asi e echnique ha can be easily
implemen ed, epo s aluable in o ma ion on he obse ed panc ea ic lesion. These p elimina y
esul s se he basis o a la ge s udy o e ine TLB se um sco e and mo e close o he clinical
applica ion o TLB p o iding use ul in o ma ion o he gas oen e ologis du ing pa ien diagnosis.
Keywo ds:
panc ea ic cys s; he mal liquid biopsy; di e en ial scanning calo ime y; diagnosis;
gene alized linea models
1. In oduc ion
Du ing ecen yea s, he de ec ion o panc ea ic cys s has become mo e equen due
o imp o emen s in abdominal imaging echniques. The incidence o his pa hology is
app oxima ely 2% in he adul popula ion [
1
]. Compu ed omog aphy (CT) scans a e e-
po ed de ec ion be ween 1.2% and 2.6%, and magne ic esonance imaging (MRI) has e en
a highe de ec ion capabili y, anging be ween 13.5% and 19.9% [
1
,
2
]. The managemen o
J. Pe s. Med. 2021,11, 25. h ps://doi.o g/10.3390/jpm11010025 h ps://www.mdpi.com/jou nal/jpm
J. Pe s. Med. 2021,11, 25 2 o 19
hese inciden ally de ec ed panc ea ic cys s is s ill a challenge, because, e en hough he
isk o being malignan is low, he p ognosis in case o panc ea ic adenoca cinoma and
in aduc al papilla y mucinous neoplasms- (IPMN)- ela ed panc ea ic adenoca cinoma
is e y poo and has no imp o ed ecen ly [
3
]. Thus, dis inguishing be ween benign
and malignan cys s is di icul , and e y o en equi es su gical in e en ion wi h con-
side able mo bidi y and mo ali y. Since 2005, some guidelines ha e been published and
upda ed: he Ame ican Socie y o Gas oin es inal Endoscopy [
4
], in e na ional consensus
guidelines by he In e na ional Associa ion o Panc ea ology (Sendai guidelines) [
5
], he
Ame ican College o Gas oen e ology [
6
], and he In e na ional Associa ion o Panc e-
a ology (Fukuoka guidelines) [
7
,
8
]. Mo e ecen ly, The Eu opean S udy G oup on Cys ic
Tumo s o he Panc eas published an upda e, eplacing he 2013 Eu opean Consensus
S a emen Guidelines [9].
F om a clinical poin o iew, he classi ica ion acco ding o he p ognosis o he lesion
is: (1) cys s wi h malignan po en ial (mucinous), (2) cys s wi hou malignan po en ial, and
(3) malignancies. Howe e , mos s udies p o ide a desc ip ion o bioma ke s o di e en i-
a ing he mucinous and non-mucinous ype o cys s. The non-mucinous g oup comp ises
se ous cys adenomas (SCAs) ( he mos common ype), panc ea ic pseudocys s (PCs), and a
a ie y o a e cys s (benign epi helial, lymphoepi helial, congeni al, and squamoid cys s).
Mos a e ound inciden ally and none o hem ep esen s a isk o becoming malignan [
10
].
On he con a y, he mucinous g oup, including mucinous cys ic neoplasms (MCNs) and
IPMNs, cons i u es he majo i y o neoplas ic p emalignan cys s iden i ied in he panc eas,
and a p ecise diagnosis echnique would be i al o he managemen o pa ien s [
11
]. The
de elopmen o echniques wi h g ea e p e-su gical diagnos ic p ecision would make
i possible o a oid he mo bidi y and mo ali y associa ed wi h a high- isk in e en ion
when i is no s ic ly necessa y, as well as educe he heal hca e o e load de i ed om
unnecessa y ou pa ien ollow-up in cys ic lesions wi hou he po en ial o malignancy.
Cys ic luid ma ke s become especially ele an when ansabdominal ul asonog-
aphy, CT o MRI a e inconclusi e. In hese cases, i is necessa y o employ ano he isk
p edic o o indica e su ge y as he mos app op ia e ea men gi en he es ima ed isk.
The p esence o amylase a high concen a ion in cys luid indica es ha he e is a
communica ion be ween he cys and he duc al sys em. This occu s in bo h pseudocys s
and IPMN lesions. When amylase le els a e lowe han 250 U/L, communica ion wi h he
conduc can be disca ded, wi h a speci ici y o 98% [
12
]. Howe e , amylase alue alone
is no enough o di e en ia e be ween mucinous/non-mucinous o MCN/IPMN, which
is impo an om he poin o iew o pa ien managemen , in deciding whe he su gical
esec ion o cys ime-moni o ing is ecommended [
13
]. A p e ious episode o panc ea i is
can be o help in dis inguishing a pseudocys om an IPMN lesion (occasionally ela ed o
panc ea i is) [14].
Ca cinoemb yonic an igen (CEA) is also employed as a bioma ke . This is a se o
highly ela ed glycop o eins in ol ed in cell adhesion, mucin being one o hem. I can
be used o dis inguish be ween cys s wi h (MCNs and IPMNs) and wi hou (SCAs and
PCs) mucinous epi helium [
10
]. The majo incon enience o CEA is he absence o an
app op ia e cu o alue [
12
]. The cu en accep ed alue o classi y a cys as mucinous is
CEA > 192 ng/mL [15].
In o de o imp o e diagnosis, s udies ela ed o he iden i ica ion o new bioma ke s
in cys ic luid o se um ha e been ecen ly epo ed [16–21].
In 2007 Chai es e al. [
22
] desc ibed he applica ion o di e en ial scanning calo ime y
(DSC) in diagnosis using plasma/se um samples om cance pa ien s. Since hen, many
s udies ha e con i med he po en ial clinical use o his echnique, no only applied o
plasma/se um [
23
–
30
], bu also o o he biological human samples such as ce eb ospinal
luid [
31
,
32
]. In 2018 ou g oup coined he name “ he mal liquid biopsy” (TLB) o DSC
applied o cance diagnosis and cance pa ien ’s ea men moni o ing [
33
,
34
]. The TLB
he mog am epo s he global dena u a ion p o ile o all he p o eins p esen in he
se um/plasma sample and he in luence o po en ial in e ac ions be ween blood plasma
J. Pe s. Med. 2021,11, 25 3 o 19
p o eins and me aboli es, he e o e e lec ing any al e a ion induced by a ce ain disease.
As in he case o plasma o se um, cys ic luid is also composed o a mix u e o p o eins
and TLB may also be applied as a clinical diagnosis ool. In his wo k, he po en ial o TLB
as a clinical bioma ke o cys classi ica ion has been pu sued. In his pilo s udy, 20 cys
luid samples we e analyzed and hei TLB cys p o iles we e ob ained, wi h he pu pose
o inding a co ela ion be ween he TLB he mog am and he ype o cys . The p o eomic
analysis also allowed a desc ip ion o he mo e abundan p o eins in he cys luid, as well
as he pos - ansla ional modi ica ions p esen in hose p o eins. TLB was also applied
o se um samples in some o he pa ien s, and TLB se um p o ile di e ences be ween
g oups was s udied o y o de e mine whe he o no di e ences obse ed in cys luid
TLB co ela ed wi h se um TLB. This would be ex emely impo an because, in case cys
ea u es a e e lec ed in ce ain se um al e a ions, a simple and isk- ee plasma/se um
TLB analysis could be employed o cys diagnosis/classi ica ion. Despi e he low numbe
o samples conside ed in his pilo s udy, di e en pa e ns in cys luid and plasma om
pa ien s wi h pa hology could be obse ed.
2. Ma e ials and Me hods
2.1. Subjec s and Samples
Cys liquid and se um samples om pa ien s wi h cys ic lesions in he panc eas de-
ec ed by ansabdominal ul asonog aphy o CT o MRI we e e e ed o he Depa men
o Diges i e Endoscopy a he Hospi al Clínico Uni e si a io Lozano Blesa (HCULB),
Za agoza, Spain, be ween Janua y 2016 and Sep embe 2018. The p ocedu e was in acco -
dance wi h he ecommenda ions o he local e hics commi ee and all pa ien s ga e hei
in o med consen . Panc ea ic cys ic luids we e collec ed by EUS-guided ine needle aspi a-
ion (FNA). The EUS-FNA p ocedu e was pe o med wi h an Olympus
®
140 cu ilinea
echo-endoscope. Bos on Scien i ic
TM
Expec
®
19 o 22-gauge needles we e used depending
on he cys ic endosonog aphic ea u es. We no ed he cha ac e is ics o he aspi a ed cys ic
luid: olume, colo , and iscosi y. The majo i y o he luid was examined by he same
cy opa hologis o e e y pa ien and he es (a leas 1 mL Eppendo o each pa ien )
was collec ed o de ec ion o biochemical ma ke s, DSC measu emen s, and p o eomic
s udies desc ibed in his manusc ip . The collec ed cys ic luid samples we e hen s o ed a
−80 ◦C un il hey we e p epa ed o analysis.
Se um samples om heal hy subjec s as con ol g oup (HC) consis ed o 85 se um sam-
ples om Spanish Caucasian subjec s, appa en ly cance - ee, om he FISABIO (Fundación
pa a el Fomen o de la In es igacion Sani a ia y Biomedica de la Comuni a Valenciana)
biobank wi h a homogeneous dis ibu ion, including gende (53% men and 47% women),
wi h an a e age age o 45.2 ±14.2.
2.2. The mal Liquid Biopsy (TLB) P o ile De e mina ion
DSC he mog ams we e measu ed using a high-sensi i i y di e en ial scanning VP-
DSC mic ocalo ime e (Mic oCal, Mal e n-Panaly ical, Mal e n, UK). Cys ic liquid sam-
ples, se um samples, and e e ence solu ions we e p ope ly degassed and ca e ully loaded
in o he cells o a oid bubble o ma ion. The baseline o he ins umen was ou inely
eco ded be o e he expe imen s. Expe imen s we e pe o med in cys ic liquid samples
(dilu ed 1:10 in phospha e bu e ed saline, PBS) and se um samples (dilu ed 1:25 in PBS) a
a scanning a e o 1
◦
C/min. The mog ams we e baseline-co ec ed and analyzed using
so wa e de eloped in ou labo a o y implemen ed in O igin 7 (O iginLab, No hamp on,
MA, USA).
2.3. Da a Analysis
We ha e de eloped a phenomenological model in which he TLB se um he mog am is
decon olu ed in o se e al indi idual ansi ions, modeling each indi idual ansi ion by he
logis ic peak o Hubbe unc ion [
30
,
33
]. This model has been success ully applied in he
analysis o se um samples om melanoma and gas ic and lung cance pa ien s [
30
,
34
,
35
].
J. Pe s. Med. 2021,11, 25 4 o 19
F om his mul ipa ame ic analysis, a TLB se um sco e (be ween 0 and 1) can be calcula ed
epo ing he le el o al e a ions in plasma (TLB se um sco e < 0.5, absence o al e a ions;
TLB se um sco e > 0.5, p esence o al e a ions).
The Kolmogo o -Smi no es was pe o med o assess he no mal dis ibu ion o he
a iables. Medians be ween wo independen g oups we e compa ed wi h he Wilcoxon
es , in non-no mal dis ibu ions. A e ages be ween wo independen g oups we e com-
pa ed wi h he - es , in no mal dis ibu ions.
2.4. P o ein Sample P epa a ion and P o ein Iden i ica ion and Quan i ica ion by
Mass Spec ome y
P o ein concen a ion: Measu ed by B ad o d p o ein assay (Bio-Rad, Mad id, Spain)
using pu i ied bo ine se um albumin (BSA) (10 mg/mL, New England BioLabs, EVRY
cedex, F ance) in PBS as s anda d. Abso bance a 595 nm o wo dilu ions om each se um
sample was measu ed in iplica e in a Syne gy HT mul imode mic opla e eade (BioTek
Ins umen s, Winooski, VT, USA).
In solu ion diges ion: Samples we e e apo a ed and esuspended in 10
µ
L o dena u -
ing bu e (6 M u ea, 100 mM T is bu e pH 7.8). Nex , cys eines we e educed wi h 1.5
µ
L
DTT (200 mM) o 30 min a 37
◦
C and alkyla ed wi h 6
µ
L o iodoace amide (200 mM) o
30 min in he da k. Un eac ed iodoace amide was consumed adding 6
µ
L o he educing
agen (200 mM DTT) o 30 min a oom empe a u e. Samples we e dilu ed wi h 50 mM
ammonium bica bona e o a u ea inal concen a ion lowe han 1 M. T ypsin diges ion
(Gold T ypsin, P omega, Madison, WI, USA) was ca ied ou o e nigh a 37
◦
C a a 1:20 en-
zyme/p o ein a io. Reac ion was s opped adding concen a ed o mic acid (Me ck KGaA,
Da ms ad , Ge many). Samples we e e apo a ed, esuspended in 2% ace oni ile (ACN),
0.1% o mic acid, and il e ed h ough 0.45 µm il e s.
P o ein iden i ica ion by LC-ESI-MS/MS: P o ein iden i ica ion was pe o med on a
nano-LC 2D sys em (LC 425, Eksigen Ekspe TM, Dublin, CA, USA) coupled o a hyb id
iple quad upole/linea ion ap mass spec ome e (4000 QTRAP, Sciex, Fos e Ci y, CA,
USA). On-line p e-concen a ion and desal ing o samples was pe o med using a C18
ap ca idge (Luna
®
0.3 mm id, 20 mm, 5
µ
m pa icle size, Phenomenex, CA, USA) a
10
µ
L/min o 5 min. Pep ide sepa a ion was pe o med using a C18 column (Gemini
®
0.3 mm id, 150 mm, 3
µ
m pa icle size, Phenomenex, CA, USA), a 5
µ
L/min o low a e.
Column was main ained a 35
◦
C. The elu ion g adien was om 5 o 35% ACN (0.1%
o mic acid) in 90 min. The mass spec ome e was in e aced wi h an ESI sou ce (Tu bo
V
™
) using a 25
µ
m ID hyb id elec ode and was ope a ed in he posi i e ion mode. MS
sou ce pa ame e s we e as ollows: capilla y ol age 5000 V, de-clus e ing po en ial (DP)
85 V and cu ain and ion sou ce gas (Ni ogen) 15 psi. Analyses we e pe o med using an
in o ma ion dependen acquisi ion (IDA) me hod wi h he ollowing s eps: single enhanced
mass spec a (EMS, 400–1400 m/z) om which he 5 mos in ense peaks we e subjec ed o
an enhanced p oduc ion [EPI (MS/MS)] scan. P o ein iden i ica ion was ca ied ou using
he Masco sea ch engine (Ma ix Science; London, UK) and he non- edundan SwissP o
da abase (553,655 sequences; 198,177,566 esidues). Sea ch pa ame e s we e monoiso opic
mass accu acy, pep ide mass ole ance
±
0.5 Da, agmen mass ole ance
±
0.3 Da; one
allowed missed clea age; allowed ixed modi ica ion ca bamido-me hyla ion (Cys), and
a iable modi ica ion oxida ion (Me ). Posi i e iden i ica ion was assigned wi h Masco
sco es abo e he h eshold le el (p< 0.05), wi h a leas wo iden i ied pep ides wi h a sco e
abo e homology
P o ein SDS elec opho esis: Samples mixed wi h NuPAGE LDS Sample bu e (In-
i ogen), and hea ed a 95
◦
C o 4 min, we e analysed by sodium dodecyl sulpha e–
polyac ylamide gel elec opho esis (SDS–PAGE) using 10% ac ylamide esol ing gels and
4% ac ylamide s acking gels (Bio-Rad). The gels we e ixed wi h a mix u e o e hanol, ace ic
acid, and deionized wa e (40:10:50) o 1 h. A e washing in wa e o 5 min, he gels
we e s ained wi h Coomassie B illian Blue R250 (0.1% in 25% me hanol, 10% ace ic acid)
and de-s ained by incuba ion in 30% ace ic acid and 20% me hanol. Molecula weigh s
we e es ima ed by compa ison wi h he mig a ion a es o s anda d p o eins (Bio-Rad).
J. Pe s. Med. 2021,11, 25 5 o 19
3. Resul s
3.1. Clinical Sample Desc ip ion
Pa ien s who unde wen endoscopic ul asonog aphy p ocedu e we e included in his
wo k. A o al o 20 subjec s, 60 and 40% men and women, espec i ely, wi h an a e age
age o 62 ±13 yea s.
Based on imaging and cy opa hology, he panc ea ic cys s we e classi ied in o di e en
ca ego ies (Table 1).
Table 1. Pa ien Desc ip ion.
Type o Cys Non-Cys
Malignan Lesions
Benign P e-Malignan
PC
(n= 5)
WOPN
(n= 3)
SC
(n= 1)
LYM
(n= 1)
IPMN
(n= 7)
MCN
(n= 1)
PDAC
(n= 2) To al (n= 20)
Age (yea s) * 63 ±10 62 ±10 72 ±0 52 ±0 72 ±13 42 ±0 40 ±2 62 ±13
Male/ emale % 80/20 67/33 100/0 0/100 71/29 0/100 0/100 60/40
* A e age
±
s anda d de ia ion (sd) PC = pseudocys ; WOPN = Walled-o panc ea ic nec osis; IPMN = in aduc al papilla y mucinous
neoplasm; SC= Se ous Cys ; MCN= Mucinous Cys adenoma; LYM= lymphocele; PDAC= Panc ea ic Duc al Adenoca cinoma.
Clinical in o ma ion o he samples is de ailed in Table 2. All he cys s we e be ween 2
and 15 cm in size and hey we e loca ed in any egion in he panc eas. Acco ding o clinical
da a (amylase and CEA concen a ions), samples we e di ided in wo g oups: benign cys s
(PC, WOPN and SC) and p emalignan cys s (IPMN and MCN). The e a e wo samples
ha u ned ou no o be cys s, bu malignan lesions (PDAC).
Table 2. Clinical Cys Sample Desc ip ion.
G oup Name Localiza ion in
he Panc eas
Cys Size
(cm)
Amylase
(U/L)
CEA
(ng/mL) Final Clinical Diagnosis
Benign Cys s
PC1 Body 5.5 >11,000.0 9.23 Pseudocys (In Acu e Panc ea i is
Con ex )
PC2 Head 4.1 >11,000.0 64.2 Pseudocys
PC3 Head 3.5 5635.0 68.4 Pseudocys
PC4 Body 15.0 nd nd Pseudocys (In Acu e Panc ea i is
Con ex )
PC5 Head 3.0 >11,000.0 28.8 Pseudocys (In Ch onic Panc ea i is
Con ex )
WOPN1 Tail 6.4 >11,000.0 2.4 Walled-o panc ea ic nec osis
WOPN2 Head 10.0 >11,000.0 2.0 Walled-o panc ea ic nec osis
WOPN3 Body 4.0 >11,000.0 50.0 Walled-o panc ea ic nec osis
SC1 Body 5.0 41.0 0.7 Se ous Cys
LYM Head 4.9 24.0 0.8 Lymphocele
P e-Malignan
Cys s
IPMN1 Body 2.6 >11,000.0 489.2 B anch duc IPMN
IPMN2 Head, Body, Tail 2.0 162.0 1488.0 Main duc IPMN
IPMN3 Head 2.3 >11,000.0 156.0 B anch duc IPMN
IPMN4 Head 2.5 >11,000.0 556.0 B anch duc IPMN
IPMN5 Is hmus 3.5 >11,000.0 225.0 Mixed B anch and Main duc IPMN
IPMN 6 Head 3.0 10.0 392.0 Main Duc IPMN wi h panc ea ic
ex ension
IPMN 7 Head, Body, Tail 3.5 4.0 >50,000.0 Main Duc IPMN wi h panc ea ic
ex ension
MCN1 Body 3.3 3401.0 1617.0 Mucinous Cys adenoma
Non-Cys
Malignan Lesions PDAC 1 Body, Tail 8.0 nd nd Panc ea ic Duc al Adenoca cinoma
PDAC 2 Head 0.5 >11,000.0 1192.0 Panc ea ic Duc al Adenoca cinoma
nd = no de e mined: Amylase < 250 U/L, communica ion wi h he conduc can be disca ded; CEA > 192 ng/mL o classi y a cys as
mucinous.
Panc ea ic pseudocys s (PC) a e pocke s o luid, common sequelae o acu e panc ea i-
is o ch onic panc ea i is. PCs a e impo an in e ms o managemen and di e en ia ion
om o he cys ic p ocesses o masses in his egion. Acco ding o he upda ed A lan a

J. Pe s. Med. 2021,11, 25 6 o 19
classi ica ion [
36
], he e a e wo main g oups o ma u e-well de ined luid collec ions asso-
cia ed wi h acu e panc ea i is: A/Fluid collec ions in in e s i ial edema ous panc ea i is
(PC), and B/Fluid collec ions in nec o izing panc ea i is (WOPN). Bo h PC and WOPN
we e conside ed benign cys s. F om ou PC samples, PC 1 and 4 we e in he con ex o
acu e panc ea i is, and PC 5 was in he con ex o ch onic panc ea i is. The WOPN cys s
had he bigges size, be ween 3 and 15 cm. Bo h ypes o panc ea ic collec ions (PC and
WOPN) we e amylase posi i e (abo e 250 U/L) and CEA nega i e (below 192 ng/mL).
Se ous cys s (SC) a e benign neoplasms composed o nume ous small cys s ha a e
a ayed in a honeycomb-like o ma ion and mos indi idual cys s a e ypically <10 mm.
Lymphocele (LYM), also known as cys ic lymphangioma, is a a e disease. The e a e
no ypical clinical mani es a ions, and mos pa ien s we e diagnosed inciden ally du ing
imaging o su ge y. The e o e, diagnosis is challenging. Su gical esec ion is s ill conside ed
as he mos e ec i e app oach o lymphocele, and p ognosis is a o able. In ou s udy, SC
and Lym we e 5 cm in size, and amylase and CEA nega i e.
In aduc al papilla y mucinous neoplasms (IPMN) a e epi helial panc ea ic cys ic
umo s o mucin-p oducing cells ha a ise om he panc ea ic duc s. They a e mos
commonly seen in elde ly pa ien s, wi h sex dis ibu ion oughly balanced, a possible
sligh male p edominance. IPMNs a e slow g owing umo s ha ha e malignan po en ial
and dis inc a ian s ha e been desc ibed: main duc (IPMN 6 and 7), b anch duc (IPMN
1, 3 and 4), and mixed b anch and main duc (IPMN 2 and 5). Main duc IPMNs ha e a
e y high a e o malignancy (up o 70% in epo ed su gical se ies [
8
]); o his eason, he
usual ecommenda ion is su gical emo al o he a ec ed po ion o he panc eas. B anch
duc IPMNs a e cys ic neoplasms o he panc eas ha ha e malignan po en ial and hei
managemen is challenging; he isk o su ge y mus be ca e ully weighed agains he isk
o malignancy when deciding on su gical emo al o su eillance. This is he eason why
g ea e o s a e aken o dis inguish mucinous cys s om o he cys lesions (specially, main
duc IPMNs). All IPMNs a e conside ed as p emalignan cys s. They had he smalles size,
be ween 2 and 3.5 cm. Fou we e amylase posi i e (abo e 250 U/L) and all we e CEA
posi i e (abo e 192 ng/mL o e y closed in case o IPMN3 wi h 156 ng/mL).
Mucinous Cys adenoma (MCN) is ano he ype o mucinous cys ic neoplasm o he
panc eas, adi ionally conside ed ypical o middle age emales. MCN1 was 3 cm in size,
amylase and CEA posi i e.
3.2. Analysis o TLB om Cys ic Liquid Samples
TLB he mog ams o 20 cys ic luid samples we e ob ained. P o ein concen a ions
and dilu ions could be conside ed, bu in his case TLB cu es we e no malized acco ding
o hei a ea unde he cu e alues (AUC); he e o e, signals om he di e en samples can
be compa ed and unce ain ies in p o ein concen a ion (inhe en o colo ime ic me hods)
a e a oided. TLB cys p o iles clus e ed acco ding o hei clinical assessmen (benign o
p emalignan na u e) a e ep esen ed in Figu e 1.
Rega ding he benign cys ic g oup, he WOPN g oup exhibi ed a e y simila cys
he mog am p o ile wi h wo peaks a 65 and 82
◦
C. We can easily dis inguish his g oup
om he o he benign cys s (Figu e 2A). PC5 is he only PC lacking he 85
◦
C peak, and i
is he only PC in a ch onic panc ea i is con ex .
In he p emalignan cys g oup, b anch duc IPMNs (IPMN1, IPMN3 and IPMN4)
exhibi ed a simila p o ile (Figu e 2C). Main duc IPMNs (IPMN6 and IPMN7) exhibi ed a
single peak.
J. Pe s. Med. 2021,11, 25 7 o 19
J. Pe s. Med. 2021, 11, x FOR PEER REVIEW 7 o 19
o hei a ea unde he cu e alues (AUC); he e o e, signals om he di e en samples
can be compa ed and unce ain ies in p o ein concen a ion (inhe en o colo ime ic
me hods) a e a oided. TLB cys p o iles clus e ed acco ding o hei clinical assessmen
(benign o p emalignan na u e) a e ep esen ed in Figu e 1.
Figu e 1. Indi idual TLB he mog ams om cys ic liquid samples. Samples we e clus e ed acco ding o hei benign o
p emalignan na u e.
Rega ding he benign cys ic g oup, he WOPN g oup exhibi ed a e y simila cys
he mog am p o ile wi h wo peaks a 65 and 82 °C. We can easily dis inguish his g oup
om he o he benign cys s (Figu e 2A). PC5 is he only PC lacking he 85 °C peak, and i
is he only PC in a ch onic panc ea i is con ex .
In he p emalignan cys g oup, b anch duc IPMNs (IPMN1, IPMN3 and IPMN4)
exhibi ed a simila p o ile (Figu e 2C). Main duc IPMNs (IPMN6 and IPMN7) exhibi ed
a single peak.
3.3. Analysis o P o eomic Signa u es om Cys ic Liquid Samples
P o eins iden i ied by LC-ESI-MS/MS (de ailed in Table S1) we e analyzed and
clus e ed acco ding o he benign o p emalignan na u e o he cys (Figu e 3). This i s
classi ica ion dis inguishes 52 p o eins common in he cys g oups, and 12 and 11 p o eins
p esen only in benign cys s and p emalignan cys s, espec i ely. A deepe analysis
acco ding o di e en g oups o cys s was pe o med.
Figu e 1.
Indi idual TLB he mog ams om cys ic liquid samples. Samples we e clus e ed acco ding o hei benign o
p emalignan na u e.
3.3. Analysis o P o eomic Signa u es om Cys ic Liquid Samples
P o eins iden i ied by LC-ESI-MS/MS (de ailed in Table S1) we e analyzed and clus-
e ed acco ding o he benign o p emalignan na u e o he cys (Figu e 3). This i s
classi ica ion dis inguishes 52 p o eins common in he cys g oups, and 12 and 11 p o-
eins p esen only in benign cys s and p emalignan cys s, espec i ely. A deepe analysis
acco ding o di e en g oups o cys s was pe o med.
3.3.1. Benign Cys s
The WOPN cys g oup exhibi ed a homogeneous p o eomic p o ile (Table S1). 18 ou
o 41 (44%) p o eins we e sha ed by all cys s in his g oup (Figu e 4A). The simila i y
in hese samples was e en highe , because 10 mo e p o eins we e common in WOPN1
and WOPN3. Low p o ein concen a ion in WOPN2 (Figu e S1A) could p e en p ope
iden i ica ion o mo e p o eins in ha sample. The mos abundan p o eins ound in his
g oup we e globulins (mac oglobulin and immunoglobulins), a ype o p o ein ela ed o
immunological esponse as a consequence o an in lamma ion p ocess. This is consis en
wi h he na u e o his speci ic ype o cys : walled-o panc ea ic nec osis (WOPN) is a
well-ci cumsc ibed a ea o nec osis which occu s as a la e complica ion o acu e panc ea i is,
gene ally a e ou weeks om he ini ial episode. Singula p o eins de ec ed in WOPN1
and WOPN3 samples we e S-100 p o eins. They belong o he S100 p o ein amily, ha ing
impo an oles in in lamma ion and may also be use ul ma ke s o gu in lamma ion [
37
].
Once sec e ed in he ex acellula space, S100A9 ac s as a chemo-a ac an , ec ui ing
J. Pe s. Med. 2021,11, 25 8 o 19
u he in lamma o y cells and c ea ing an in lamma o y mic oen i onmen ha p omo es
umo de elopmen [38].
J. Pe s. Med. 2021, 11, x FOR PEER REVIEW 8 o 19
Figu e 2. TLB he mog ams om cys ic liquid samples clus e ed acco ding o hei ype. A e age
cu es (colo ed lines) and s anda d de ia ions o cu e alues (g ey) a e ep esen ed: WOPNs (A),
PCs (B), IPMNs (C) and IPMN7/MCNs (D).
Figu e 3. LC-ESI-MS/MS p o eomic con en o cys ic samples. Common p o eins we e analyzed ia Venn diag ams online
ool (h p://bioin o ma ics.psb.ugen .be/beg/ ools/ enn-diag ams). 12 and 11 p o eins we e de ec ed only in benign cys s
(blue) and p emalignan cys s (pink), espec i ely, and 52 p o eins appea ed in bo h g oups (in e sec ion se ). Table
comp ises he de ailed in o ma ion o he p o eins in each se .
52
1112
PREMALIGNANT
CYSTS
BENIGN
CYSTS
Figu e 2.
TLB he mog ams om cys ic liquid samples clus e ed acco ding o hei ype. A e age
cu es (colo ed lines) and s anda d de ia ions o cu e alues (g ey) a e ep esen ed: WOPNs (
A
),
PCs (B), IPMNs (C) and IPMN7/MCNs (D).
J. Pe s. Med. 2021, 11, x FOR PEER REVIEW 8 o 20
Figu e 2. TLB he mog ams om cys ic liquid samples clus e ed acco ding o hei ype. A e age
cu es (colo ed lines) and s anda d de ia ions o cu e alues (g ey) a e ep esen ed: WOPNs (A),
PCs (B), IPMNs (C) and IPMN7/MCNs (D).
Figu e 3. LC-ESI-MS/MS p o eomic con en o cys ic samples. Common p o eins we e analyzed ia Venn diag ams online
ool (h p://bioin o ma ics.psb.ugen .be/beg/ ools/ enn-diag ams). 12 and 11 p o eins we e de ec ed only in benign cys s
(blue) and p emalignan cys s (pink), espec i ely, and 52 p o eins appea ed in bo h g oups (in e sec ion se ). Table com-
p ises he de ailed in o ma ion o he p o eins in each se .
52
1112
PREMALIGNANT
CYSTS
BENIGN
CYSTS
Figu e 3.
LC-ESI-MS/MS p o eomic con en o cys ic samples. Common p o eins we e analyzed ia Venn diag ams online
ool (h p://bioin o ma ics.psb.ugen .be/beg/ ools/ enn-diag ams). 12 and 11 p o eins we e de ec ed only in benign
cys s (blue) and p emalignan cys s (pink), espec i ely, and 52 p o eins appea ed in bo h g oups (in e sec ion se ). Table
comp ises he de ailed in o ma ion o he p o eins in each se .
J. Pe s. Med. 2021,11, 25 9 o 19
J. Pe s. Med. 2021, 11, x FOR PEER REVIEW 10 o 19
Figu e 4. LC-ESI-MS/MS p o eomic con en o cys ic samples acco ding o cys ic ypes: (A) WOPN, (B) PC, (C) IPMN, and (D)
MCN+IPMN. Common p o eins we e analyzed ia Venn diag ams online ool. Colo s code o di e en g oups and numbe s
inside each se and sha ed sub-se s indica e he numbe o iden i ied p o eins.
BENIGN CYSTS
A B
WOPN1,2,3 WOPN1,3
PC2,3,
4
,5
P
0
27
6
3
-
A
l
p
ha-1-ac
i
d g
l
y
co
p
o e
i
n 1
P01023-Alpha-2-mac oglobulin
P01023-Alpha-2-mac oglobulin
P04746-Panc ea ic alpha-amylase
P01024-Complemen C3
P69905-Hemoglobin subuni alpha
P68871-Hemoglobin subuni be a
P02042-Hemoglobin subuni del a
P02790-Hemopexin
P00738-Hap oglobin
P0DOX2-Immunoglobulin alpha-2 hea y chain
P0DOX5-Immunoglobulin gamma-1 hea y chain
P01876-Immunoglobulin hea y cons an alpha 1
P01859-Immunoglobulin hea y cons an gamma 2
P01860-Immunoglobulin hea y cons an gamma 3
P01861-Immunoglobulin hea y cons an gamma 4
P01834-Immunoglobulin kappa cons an
P02787-Se o ans e in
P59
6
6
5
-
Neu o
p
h
i
l
de
ens
i
n 1
P02679-Fib inogen gamma chain
P55259-Panc ea ic sec e o y g anule
memb ane majo glycop o einGP2
P02100-Hemoglobin subuni epsilon
P01857-I mmunoglobulin hea y cons an
gamma 1
P01871-Immunoglobulin hea y cons an mu
P0DOY2-I mmunoglobulin lambda cons an 2
P16233-Panc ea ic iacylglyce ol lipase
P05109-P o ein S100-A8
P06702-P o ein S100-A9
P
0
4
7
4
6
-
Panc ea
i
c a
l
p
ha-am
y
l
ase
P08861-Chymo ypsin-like elas ase amily membe 3B
P15086-Ca boxypep idase B
P15085-Ca boxypep idase A1
P09093-Chymo ypsin-like elas ase amily membe 3A
PC2,3,5
P
4
8
0
52
-
C
a
b
ox
y
p
e
p
i
dase A2
P08217-Chymo ypsin-like elas ase amily membe 2A
Q99895-Chymo ypsin-C
P16233-Panc ea ic iac ylgly ce ol lipase
P54317-Panc ea ic lipase- ela ed p o ein 2
P07477-T ypsin-1
PC3,
4
,5
P
0
27
6
8
-
A
l
b
um
i
n
P04745-Alpha-amylase 1A
P68871-Hemoglobin subuni be a
P05451-Li hos a hine-1-alpha
PRE-MALIGNANT CYSTS
C D
IPMN 1,2,3,4,6,7
IPMN 2,3,4
B anc h and Main Duc +
MCN1
B anc h and Main Duc
bu no in MCN1
Main Duc + MCN1
P
0
27
6
8
-
A
l
b
um
i
n
P04745-Alpha-amylase 1A
P19961-Alpha-amylase 2B
P04746-Panc ea ic alpha-
amylase
P15085-Ca boxypep idase A1
P48052-Ca boxypep idase A2
P15086-Ca boxypep idase B
P08217-Chymo ypsin-lik e
elas ase amily membe 2A
P09093-Chymo ypsin-lik e
elas ase amily membe 3A
P08861-Chymo ypsin-lik e
elas ase amily membe 3B
P04118-Colipase
P17538-Chymo ypsinogen B
Q99895-Chymo ypsin-C
P68871-Hemoglobin subuni
be a
P
6
99
0
5
-
Hemog
l
o
b
i
n
subuni alpha
P02042-Hemoglobin
subuni del a
P02100-Hemoglobin
subuni epsilon
P69891-Hemoglobin
subuni gamma-1
P01876-
I mmunoglobulin hea y
cons an alpha 1
P0DOX7-
I mmunoglobulin kappa
ligh chainI GLC2_HUM
P00995-Se ine p o ease
inhibi o Kazal- ype 1
P16233-Panc ea ic
iac ylgly ce ol lipase
P61626-Lysozyme C
P98088-Mucin-5AC
Q9HC84-Mucin-5B
P05451-Li hos a hine-1-
alpha
P07477-T ypsin-1
P
0
4
7
4
6
-
Panc ea
i
c a
l
p
ha-
amylase
P04745-Alpha-amylase 1A
P19961-Alpha-amylase 2B
P15086-Ca boxypep idase
B
P08217-Chymo ypsin-like
elas ase amily membe 2A
P09093-Chymo ypsin-like
elas ase amily membe 3A
P69905-Hemoglobin
subuni alpha
P
0
27
6
8
-
A
l
b
um
i
n
P69905-Hemoglobin
subuni alpha
P69905-Hemoglobin
subuni alpha
P02042-Hemoglobin
subuni del a
P02100-Hemoglobin
subuni epsilon
P69891-Hemoglobin
subuni gamma-1
P01876-Immunoglobulin
hea y cons an alpha 1
P61626-Lysozyme C
P98088-Mucin-5AC
Q9HC84-Mucin-5B
P0DOY2-I mmunoglobulin
lambda cons an 2
P
0
4
7
4
5
-
A
l
p
ha-am
y
l
ase 1A
P19961-Alpha-amylase 2B
P04746-Panc ea ic alpha-amylase
P15085-Ca boxypep idase A1
CBPA2_HUM
P15086-Ca boxypep idase B
P08217-Chymo ypsin-lik e elas ase
amily membe 2A
P09093-Chymo ypsin-lik e elas ase
amily membe 3A
P08861-Chymo ypsin-lik e elas ase
amily membe 3B
P0DOX7-Immunoglobulin kappa
ligh chain
P00995-Se ine p o ease inhibi o
Kazal- ype 1
P16233-Panc ea ic iacylglyce ol
lipase
P05451-Li hos a hine-1-alpha
P07477-T ypsin-1
P04118-Colipase
P17538-Chymo ypsinogen B
Q99895-Chymo ypsin-C
P19
6
52
-
A
l
p
ha-1-ac
i
d
glycop o ein 2
P02647-Apolipop o ein A-I
P00915-Ca bonic anhyd ase 1
P01024-Complemen C3
P59665-Neu ophil de ensin 1
P02790-Hemopexin
P00738-Hap oglobinI
P0DOX5-Immunoglobulin
gamma-1 hea y chain
P01859-Immunoglobulin hea y
cons an gamma 2
P01860-Immunoglobulin hea y
cons an gamma 3
P01861-Immunoglobulin hea y
cons an gamma 4
P01871-Immunoglobulin hea y
cons an mu
P01834-Immunoglobulin kappa
cons an
Q08380-Galec in-3-binding
p o ein
P80188-Neu ophil gela inase-
associa ed lipocalin
Q6UX06-Ol ac omedin-4
P01833-Polyme ic
immunoglobulin ecep o
P02787-Se o ans e in
P02774-Vi amin D-binding
p o ein
IPMN 2,6,7
P
0
27
6
8
-
A
l
b
um
i
n
P01876-Immunoglobulin
hea y cons an alpha 1
Figu e 4.
LC-ESI-MS/MS p o eomic con en o cys ic samples acco ding o cys ic ypes: (
A
) WOPN, (
B
) PC, (
C
) IPMN, and
(
D
) MCN+IPMN. Common p o eins we e analyzed ia Venn diag ams online ool. Colo s code o di e en g oups and
numbe s inside each se and sha ed sub-se s indica e he numbe o iden i ied p o eins.
J. Pe s. Med. 2021,11, 25 16 o 19
mul ipa ame ic analysis [
30
] and, la e , a TLB se um sco e [
33
] o manage and assess TLB
he mog ams acco ding o a simple in e p e a ion index (TLB se um sco e om 0 o 1) ha
could be implemen ed in diagnosis, easily allowing he s a i ica ion o he pa ien s.
We i s compa ed he TLB se um he mog ams om pa ien s o he mog ams om
heal hy subjec s ha we e no su e ing om he disease. We applied a gene al TLB se um
sco e p e iously epo ed [
33
] and he esul s we e s a is ically di e en when compa ing
heal hy subjec s wi h benign o malignan cys pa ien s, indi idually o oge he as a pooled
g oup o pa ien s (p- alues lowe han 0.05) (Figu e 6). Speci ici y and sensi i i y alues
(98% and 71%, espec i ely), as well as PPV and NPV alues (83% and 98%, espec i ely),
we e qui e high.
TLB se um sco es ( om benign o p emalignan cys g oups) we e no s a is ically
di e en when using he TLB gene al o mula when compa ing o heal hy subjec s. The e-
o e, on ha basis i was no possible o dis inguish be ween bo h ypes o cys . The e o e,
we ocused ou a en ion on speci ically compa ing bo h pa ien s’ g oups and ob aining a
cys -speci ic TLB se um sco e ha could be applied o e alua e in a-g oup cys pa ien
a iabili y. This new TLB se um sco e would s eng hen he disc imina ion powe , based on
he speci ic pa ame e s e lec ing di e ences be ween cys s. The challenge is conside able,
because a mono- a ian analysis o cys TLB pa ame e s (Table S3) showed ha none o he
indi idual pa ame e s was s a is ically di e en be ween he benign and p emalignan cys
g oup. As we p e iously con i med in ou s udies, he combina ion o all he pa ame e s
in a mul ipa ame ic-based single TLB se um sco e inc eased he disc imina ion abili y.
Despi e he small pa ien sample coho , i was possible o dis inguish be ween benign
and p emalignan cys s. This speci ic TLB se um sco e ( alues om 0 o 1) we e o e
0.75 (acco ding o Youden) in 6 ou o 8 (75%) p emalignan samples, while TLB se um
sco es we e below 0.75 in 6 ou o 6 (100%) benign samples. The diagnosis accu acy based
in he a ea unde he cu e (AUC) was 0.875, a p omising s a ing poin o ex ending
he s udy. The e o e, mo e se um samples om pa ien s wi h panc ea ic cys s should be
included in a la ge u u e s udy, bu hese p elimina y esul s a e p omising and allow us
o o esee ha he TLB se um sco e could be applied ou inely in he clinic as an addi ional
complemen a y ool helping physicians in making be e diagnos ic decisions.
5. Conclusions
TLB analysis can be applied o bo h plasma ic se um and cys luid as a so o
high in o ma ion con en ool. Despi e he small numbe o samples in his pilo s udy,
i ep esen s a p oo o concep o de eloping a use ul echnique o classi ying and
e alua ing isk in panc ea ic cys s based on liquid biopsy in bo h body luids. A u u e,
la ge s udy, wi h a la ge numbe o samples o each cys ca ego y, could con i m whe he
TLB o plasma o cys luid could be a new diagnosis ool o di e en ia e be ween benign
and p emalignan panc ea ic cys s o help clinician gas oen e ologis s in making decisions
abou disease managemen . In case o se um, TLB would ep esen a quick, low- isk,
minimally in asi e ool easily ansla ed o clinical p ac ice o diagnosis and pa ien
moni o ing. In addi ion, TLB is easonably cheap o se um es s (an es ima ed cos o
100–200
€
/$ pe es , al hough wi h a highe cos o cys luid es ), and could be pe o med
wi h p ede ined equency o pa ien su eillance.
Supplemen a y Ma e ials:
The ollowing a e a ailable online a h ps://www.mdpi.com/2075-442
6/11/1/25/s1, Figu e S1: Elec opho esis analysis o p o eomic p o iles, Figu e S2: LC-ESI-MS/MS
p o eomic con en o cys samples ypes iden i ica ion, Figu e S3: ROC cu e illus a ing he s a is ical
pe o mance o TLB se um sco e (heal hy s. cys s); Table S1: De ailed in o ma ion o cys p o eomic
p o iles, Table S2: Mono- a ian analysis o TLB pa ame e s o heal hy con ols and cys s pa ien s,
Table S3: Mono- a ian analysis o TLB pa ame e s o benign and p emalignan cys s pa ien s.

J. Pe s. Med. 2021,11, 25 17 o 19
Au ho Con ibu ions:
Concep ualiza ion, A.V.-C., and O.A.; me hodology, S.H.-D., J.L.O., O.S.-G.,
A.V.-C., and O.A.; so wa e, S.H.-D., J.L.O., O.S.-G., A.V.-C., and O.A.; alida ion, J.L.O., S.V., O.S.-G.,
A.V.-C., and O.A.; o mal analysis, J.L.O., S.V., O.S.-G., O.A., and A.V.-C.; in es iga ion, L.C, S.V.,
A.V.-C., and O.A.; esou ces, G.G.-R., J.L.O., O.S.-G., Á.L., C.S., A.V.-C., and O.A.; da a cu a ion,
S.H.-D., G.G.-R., C.S., J.L.O., and O.A.; w i ing—o iginal d a p epa a ion, S.H.-D., L.C.-L., J.L.O.,
A.V.-C., and O.A.; w i ing— e iew and edi ing, S.H.-D., G.G.-R., L.C.-L., J.L.O., S.V., O.S.-G., Á.L.,
J.M., A.V.-C., and O.A.; isualiza ion, J.L.O., L.C.-L., O.S.-G., A.V.-C., and O.A.; supe ision, Á.L.,
A.V.-C., and O.A.; p ojec adminis a ion, O.A. and A.V.-C.; unding acquisi ion, Á.L., O.A., and
A.V.-C. All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding:
This esea ch was unded by he Spanish Minis y o Economy and Compe i i eness and
Eu opean ERDF Funds (MCIU/AEI/FEDER, EU) (BFU2016-78232-P o A.V.C.); P ojec s unded by
Ins i u o de Salud Ca los III and co- unded by Eu opean Union (ESF, ”In es ing in you u u e”):
“PI15/00663 (FIS p ojec o O.A)”, “PI18/00349 (FIS p ojec o O.A. and Con ac o LC)”, “FI19/00146
(PFIS con ac o SHD)”, “CPII13/00017 (Miguel Se e P og am o OA)”; Dipu ación Gene al de
A agón (P o ein Ta ge s and Bioac i e Compounds G oup E45_17R o A.V.C. and Diges i e Pa hology
G oup B25_17R o O.A.); and he Cen o de In es igación Biomédica en Red en En e medades
Hepá icas y Diges i as (CIBERehd).
Ins i u ional Re iew Boa d S a emen :
The s udy was conduc ed in acco dance wi h he Decla a ion
o Helsinki, and he p o ocol was app o ed by he E hics Commi ee o CEICA (PI16/0228).
In o med Consen S a emen :
All subjec s ga e hei in o med consen o inclusion be o e hey
pa icipa ed in he s udy.
Da a A ailabili y S a emen :
The da a p esen ed in his s udy a e a ailable on eques om he
co esponding au ho .
Acknowledgmen s:
P o eomic analyses we e pe o med in he P o eomics Pla o m o Se icios
Cien í ico Técnicos del CIBA (IACS-Uni e sidad de Za agoza), P o eoRed ISCIII membe , Za agoza,
Spain.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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