cell-09-701328 June 4, 2021 Time: 12:12 # 1
ORIGINAL RESEARCH
published: 07 June 2021
doi: 10.3389/ cell.2021.701328
Edi ed by:
Milica Peši ´
c,
Uni e si y o Belg ade, Se bia
Re iewed by:
Ul ike Resch,
Medical Uni e si y o Vienna, Aus ia
Ka men S anko ,
Uni e si y o No i Sad, Se bia
*Co espondence:
Ana Bela Sa men o-Ribei o
[email p o ec ed]
Special y sec ion:
This a icle was submi ed o
Cellula Biochemis y,
a sec ion o he jou nal
F on ie s in Cell and De elopmen al
Biology
Recei ed: 27 Ap il 2021
Accep ed: 13 May 2021
Published: 07 June 2021
Ci a ion:
Gonçal es AC, Al es R,
Baldei as I, Jo ge J, Ma ques B,
Pai a A, Oli ei os B, Co esão E,
Nascimen o Cos a JM and
Sa men o-Ribei o AB (2021) Oxida i e
S ess Pa ame e s Can P edic he
Response o
E y h opoiesis-S imula ing Agen s in
Myelodysplas ic Synd ome Pa ien s.
F on . Cell De . Biol. 9:701328.
doi: 10.3389/ cell.2021.701328
Oxida i e S ess Pa ame e s Can
P edic he Response o
E y h opoiesis-S imula ing Agen s in
Myelodysplas ic Synd ome Pa ien s
Ana C is ina Gonçal es1,2,3,4, Raquel Al es1,2,3,4, Inês Baldei as5,6, Joana Jo ge1,2,3,4,
Bá ba a Ma ques1,7, A u Pai a2,3,8, Bá ba a Oli ei os5, Emília Co esão1,2,3,4,7,
José Manuel Nascimen o Cos a2,3,9 and Ana Bela Sa men o-Ribei o1,2,3,4,7*
1Uni e si y o Coimb a, Labo a o y o Oncobiology and Hema ology and Uni e si y Clinic o Hema ology, Facul y o Medicine
(FMUC), Coimb a, Po ugal, 2Uni e si y o Coimb a, G oup o En i onmen , Gene ics and Oncobiology (CIMAGO) – Ins i u e
o Clinical and Biomedical Resea ch (iCBR), FMUC, Coimb a, Po ugal, 3Uni e si y o Coimb a, Cen e o Inno a i e
Biomedicine and Bio echnology (CIBB), Coimb a, Po ugal, 4Clinical Academic Cen e o Coimb a (CACC), Coimb a,
Po ugal, 5Uni e si y o Coimb a, FMUC, Coimb a, Po ugal, 6Uni e si y o Coimb a, Cen e o Neu oscience and Cell
Biology, Coimb a, Po ugal, 7Clinical Hema ology Depa men , Cen o Hospi ala e Uni e si á io de Coimb a (CHUC),
Coimb a, Po ugal, 8Cen o Hospi ala e Uni e si á io de Coimb a (CHUC), Unidade de Ges ão Ope acional em Ci ome ia,
Coimb a, Po ugal, 9Uni e si y o Coimb a, Uni e si y Clinic o Oncology, FMUC, Coimb a, Po ugal
Oxida i e s ess has been implica ed in he de elopmen o se e al ypes o cance ,
including myelodysplas ic synd omes (MDS), as well as in he esis ance o ea men .
In his wo k, we assessed he po en ial o oxida i e s ess pa ame e s o p edic he
esponse o e y h opoiesis-s imula ing agen s (ESAs) in lowe - isk MDS pa ien s. To his
end, we analyzed he sys emic le els o eac i e species (pe oxides and NO), an ioxidan
de enses (u ic acid, i amin E, i amin A, GSH, GSSG, TAS, as well as GPX and GR
ac i i ies], and oxida i e damage (8-OH-dG and MDA) in 66 MDS pa ien s, om hose
44 ha e been ea ed wi h ESA. We also calcula ed he pe oxides/TAS and NO/TAS
a ios and analyzed he gene exp ession o le els o he edox egula o s, NFE2L2 and
KEAP1. We ound ha pa ien s ha espond o ESA ea men showed lowe le els
o plasma pe oxides (p<0.001), cellula GSH (p<0.001), and cellula GR ac i i y
(p= 0.001) when compa ed o pa ien s who did no espond o ESA ea men . ESA
esponde s also showed lowe le els o pe oxides/TAS a io (p<0.001) and highe
le els o he exp ession o he NFE2L2 gene (p= 0.001) han hose ha did no espond
o ESA ea men . The le els o plasma ic pe oxides shown o be he mos accu a e
bioma ke o ESA esponse, wi h good sensi i i y (80%) and speci ici y (100%) and
is an independen bioma ke associa ed wi h he apy esponse. O e all, he p esen
s udy demons a ed a co ela ion be ween oxida i e s ess le els and he esponse o
ESA ea men in lowe - isk MDS pa ien s, wi h he plasma ic pe oxides le els a good
p edic i e bioma ke o d ug (ESA) esponse.
Keywo ds: myelodysplas ic synd ome, e y h opoiesis-s imula ing agen s, esponse bioma ke , oxida i e s ess,
an ioxidan de enses, eac i e oxygen species
F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g 1June 2021 | Volume 9 | A icle 701328
cell-09-701328 June 4, 2021 Time: 12:12 # 2
Gonçal es e al. Oxida i e S ess as ESA-Response Bioma ke
INTRODUCTION
Myelodysplas ic synd omes (MDS) a e clonal hema ological
malignancies ha comp ises se e al sub ypes wi h di e en
biological and clinical p esen a ions. These diseases a e
equen ly cha ac e ized by ine icien hema opoiesis, dysplasia
in one o mo e myeloid cell lineages, a iable deg ees and
numbe o cy openias, and inc eased isk o p og ession o
acu e myeloid leukemia (AML) (Jhanwa , 2015;Cazzola,
2020). MDS blood show ypical mo phological ea u es such
as dysplasia, di e en ia ion a es , de ec i e cellula unc ions,
and genomic ins abili y. The pe iphe al cy openias equen ly
obse ed in hese pa ien s a e a consequence o he ine ec i e
hema opoiesis and could in ol e all myeloid lineages (Mu i,
2004). One o he MDS pa adoxes is he coexis ence o pe iphe al
cy openias wi h hype plas ic bone ma ow. The MDS clones
exhibi inc eased p oli e a ion, howe e , his p oli e a ion is
apidly balanced by inc eased apop osis, namely in low isk
MDS sub ypes. Recen ly, p ecu so s condi ions o MDS, such
as clonal hema opoiesis o inde e mina e po en ial (CHIP) and
clonal cy openia o unde e mined signi icance (CCUS), has been
iden i ied (Cazzola, 2020) ha allow an ea lie diagnosis and
could modi y he MDS app oach.
The p esence o cy openia and dysplasia, in a leas one
hema ologic cell line, a e essen ial o MDS diagnosis (Cazzola,
2020). On he o he hand, he isk o dea h om cy openias
complica ions o e olu ion o AML is highly a iable in MDS
pa ien s. The Re ised In e na ional P ognos ic Sco ing Sys em
(IPSS-R) is he mos equen ly used p ognos ic sys em and is
based on a small numbe o ea u es wi h independen p ognos ic
alue, including ch omosomal abno mali ies, bone ma ow
blas s, hemoglobin le el, pla ele coun and absolu e neu ophil
coun . These ea u es a e ou inely a ailable in all clinical cen e s
and allow he classi ica ion o MDS pa ien s in i e isk subg oups
( e y low isk, low isk; in e media e, high isk, and e y high
isk) wi h di e en p obabili ies o AML p og ession and su i al
(Ga cia-Mane o, 2010;Adès e al., 2014;Cazzola, 2020). In
clinical p ac ice, clinicians usually dis inguish pa ien s in lowe -
isk MDS (IPSS-R isk sco e ≤3.5) and in highe - isk MDS
(IPSS-R isk sco e >3.5), and lowe - isk MDS accoun o abou
wo- hi ds o all MDS cases (Kom okji e al., 2011;Cazzola,
2020). In lowe - isk MDS pa ien s wi h symp oma ic anemia he
i s -line ea men op ion is e y h opoiesis-s imula ing agen s
(ESAs), namely ecombinan human e y h opoie ins ( HuEPOs),
alone o combined wi h g anulocy e colony-s imula ing ac o
(G-CSF), which could p e en o delay ans usion dependency
and imp o e quali y o li e. Howe e , hese ea men s a e only
ecommended o pa ien s wi h se um e y h opoie in (sEPO)
le els below 500 U/L, and he mos eliable p edic o o a
esponse is a sEPO lowe han 200 U/L, while a high ans usion
equi emen p edic s ea men ailu e (Cazzola, 2020;Pa k e al.,
2020). Howe e , in clinical p ac ice, a sEPO cu o le el o
200 U/L is mo e indica i e o esponse (Pa k e al., 2020).
New bioma ke s and sco ing sys ems should be in es iga ed o
imp o e esponse and/o o p edic esis ance o ESA he apy.
Reac i e oxygen species (ROS) a e ele an playe s in
hema ological malignancies de elopmen , p og ession, and
he apy esis ance (Sa dina e al., 2012;Sa men o-Ribei o e al.,
2012;Gonçal es e al., 2015). These ee adical molecules show
bo h bene icial and dele e ious consequences (Gha a i, 2008;
Imbesi e al., 2013). When ROS le els o e come he cellula
an ioxidan de enses, oxida i e s ess is es ablished as esul o
an imbalance in edox homeos asis (Gha a i, 2008;Sa dina
e al., 2012). Se e al signaling pa hways such as p oli e a ion,
di e en ia ion, and apop osis a e egula ed by in acellula ROS
le els (Imbesi e al., 2013;Hasselbalch e al., 2014). Blood
cells om MDS pa ien s show inc eased le els o ROS and
dec eased concen a ion o GSH (Gho i e al., 2007;Gonçal es
e al., 2015). Fu he mo e, he dis up ion o edox homeos asis
is a c ucial ac o in d ug esis ance de elopmen , which is an
impo an ac o in he ailu e o an icance (Liu e al., 2016).
Du ing e y h oid di e en ia ion, e y h oblas s a e exposed o an
oxida i e en i onmen and ROS a e p oduced in esponse o
EPO (Zhao e al., 2016;Beneduce e al., 2019). These ROS could
ac as second messenge s by modula ing in acellula signaling
kinases including JAK2, LYN, and FYN (Beneduce e al., 2019).
Beneduce e al. (2019) ound ha in he absence o FYN kinase,
he e iciency o he EPO signal is dec eased and an inc ease in
ROS p oduc ion is obse ed (Beneduce e al., 2019). Howe e , he
dynamics o oxida i e s a us du ing e y h opoiesis and e y h oid
di e en ia ion in esponse o EPO a e s ill unclea . Addi ionally,
he nuclea ac o e y h oid 2- ela ed ac o 2 (NRF2), a majo
cellula edox modula o , plays a c ucial ole in p e en ing cance
cells om cy o oxici y induced by chemo he apy, con ibu ing
o d ug esis ance and he apeu ic ailu e. In highe - isk MDS
pa ien s, NRF2 exp ession showed a signi ican p ognos ic alue
o o e all su i al being associa ed wi h cy a abine esis ance
(Lin e al., 2019). In his con ex , we in es iga e he po en ial o
oxida i e s ess pa ame e s as p edic i e bioma ke s o esponse
o ESA ea men in MDS pa ien s.
MATERIALS AND METHODS
S udy Popula ion
This s udy en olled 66 MDS pa ien s a diagnosis om Oc obe
2012 o Ma ch 2014. MDS pa ien s we e diagnosed acco ding
o he Wo ld Heal h O ganiza ion 2016 classi ica ion o myeloid
neoplasms (A be e al., 2016) in he ollowing sub ypes: MDS
wi h single lineage dysplasia (MDS-SLD), MDS wi h mul ilineage
dysplasia (MDS-MD), MDS wi h ing side oblas s (MDS-RS),
and MDS wi h excess blas s (MDS-EB). Pa ien s we e s a i ied
acco ding o IPSS-R in lowe isk and highe - isk pa ien s
(Cazzola, 2020). Biodemog aphic (age and gende ) and clinical
da a, when a ailable, we e ob ained om medical eco ds.
The E hics Commi ee o he Facul y o Medicine o
he Uni e si y o Coimb a (Coimb a, Po ugal) app o ed he
esea ch p ocedu es, and he s udy was conduc ed ollowing he
Decla a ion o Helsinki. Be o e en ollmen , pa icipan s p o ided
hei in o med consen o pa icipa ion. The in e na ional
e hical guidelines o con iden iali y, anonymi y o pe sonal
da a, and abandonmen op ion, in case o exp essed, will
we e be ollowed.
F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g 2June 2021 | Volume 9 | A icle 701328
cell-09-701328 June 4, 2021 Time: 12:12 # 3
Gonçal es e al. Oxida i e S ess as ESA-Response Bioma ke
Sample P epa a ion
Pe iphe al blood samples we e collec ed a diagnosis, a e
as ing, in o sodium hepa in ubes. Samples we e immedia ely
cen i uged; plasma and ed blood cells (wi h a concen a ion
o hemoglobin adjus ed a 100 g/l) we e s o ed ozen a -
20◦C un il analysis, as p e iously desc ibed (Baldei as e al.,
2010). Some oxida i e s ess pa ame e s we e no malized o o al
plasma choles e ol.
U ic Acid De e mina ions
Plasma ic le els o u ic acid we e de e mined by a colo ime ic
me hod (Ba ham and T inde , 1972) based on he educ ion
o u ic acid by he u icase enzyme. In his eac ion, hyd ogen
pe oxide is eleased and o ms a ch omogenic compound
e alua ed spec opho ome ically a 550 nm.
Vi amin A and E Measu emen s
The plasma ic le els o i amins A ( i A) and E ( i E)
we e assessed in lipid ex ac s ob ained om plasma samples
and quan i ied by high-pe o mance liquid ch oma og aphy
(HPLC) using an analy ic column sphe iso b ODS1-5 µm
(250 mm ×4.6 mm), elu ed a 2.5 ml/min wi h a wa e
solu ion o me hanol (90%), a 45◦C, and de ec ed in a
spec opho ome e (Gilson) a 340 nm ( o i A) o 295 nm
( o i E). The e y h ocy ic i amin E con en was ex ac ed
in n-hexane and quan i ied by e e se-phase HPLC (Va asse y
e al., 1978;De Leenhee e al., 1979), using an analy ic column
sphe iso b S10w (250 mm ×4.6 mm), elu ed a 1.5 ml/min
wi h n-hexane modi ied wi h 0.9% o me hanol, and de ec ed by
spec opho ome y a 287 nm (Gilson).
Oxidized and Reduced Glu a hione
Quan i ica ion
The e y h ocy ic educed glu a hione (GSH) and oxidized
glu a hione (GSSG) we e also e alua ed by HPLC wi h
luo ime ic de ec ion (exci a ion a 385 nm, and emission
a 515 nm), using he Immunodiagnos ik ki (Immunodiagnos ik
AG, Bensheim, Ge many), as desc ibed by he manu ac u e .
An ioxidan Enzymes Ac i i y
De e mina ion
The glu a hione pe oxidase (GPX) ac i i y in ed blood
cells was e alua ed by spec opho ome y using an indi ec
de e mina ion me hod and e -bu yl hyd ope oxide as subs a e
(Paglia and Valen ine, 1967). The GSSG o ma ion was
examined h ough he quan i ica ion o educed nico inamide
adenine dinucleo ide phospha e (NADPH) oxida ion a
340 nm in a he mos a ed spec opho ome e UVIKON 933
UV/Visible. The ac i i y o glu a hione educ ase (GR) in ed
blood cells was e alua ed by spec opho ome y a 340 nm
(Goldbe g and Spoone , 1983), using GSSG as a subs a e, and
moni o ing i s educ ion o GSH h ough he assessmen o
NADPH oxida ion a 37◦C in a spec opho ome e UVIKON
933 UV/Visible.
Plasma ic Pe oxide Quan i ica ion
The plasma ic le els o pe oxides we e quan i ied using he ki
The mo Scien i ic Pie ce Quan i a i e Pe oxide Assay Ki – lipid-
compa ible o mula ion (Li e Technologies), acco ding o he
manu ac u e , in a Syne gyTM mul i-mode mic opla e eade
(BioTek Ins umen s).
To al An ioxidan S a us E alua ion
The plasma ic o al an ioxidan s a us (TAS) was assessed by a
ch omogenic me hod (Randox Labo a o ies) based on he plasma
capaci y o inhibi he o ma ion o he ABTS+ adical ca ion
(2,20-azino-di-[3-e ilbenzo iazolin sul ona e]), and de ec ed a
600 nm as desc ibed by he manu ac u e .
Lipid Pe oxida ion Measu emen s
The plasma ic and e y h ocy ic lipid pe oxida ion we e assessed
by he o ma ion o hioba bi u ic acid (TBA) adduc s o
malondialdehyde (MDA), sepa a ed by HPLC (Gilson), and
quan i ied luo ime ically using he ClinRep comple e ki
(RECIPE), as desc ibed by he manu ac u e . B ie ly, 100 µl
blank, s anda d, con ols, and pa ien s’ samples we e i s
de i a ized a 100◦C o 60 min in a glass ligh -p o ec ed
ial. A e cooling, samples we e neu alized, p ecipi a ed, and
cen i uged a 10,000 g o 5 min. Finally, 20 µl o he
supe na an s we e injec ed in o he HPLC and he MDA adduc s
we e de e mined luo ime ically (exci a ion a 515 nm, and
emission a 553 nm; FP-2020/2025, Jasco, Tokyo, Japan).
Plasma ic Ni ic Oxide Quan i ica ion
The plasma ic le els o ni ic oxide (NO) we e de e mined
by a pho ome ic me hod (Roche Diagnos ics GmbH) based
on he de ec ion o i s oxida ion p oduc s, ni i e and ni a e
(Ti he adge, 1998). Fi s , he ni a e p esen in he ul a-
il a ed plasma was educed o ni i e, which hen eac ed
wi h sulphanilamide and N-(1-naph hyl)-e hylenediamine
dihyd ochlo ide o gi e a ed- iole diazo dye, de ec ed by
spec opho ome y a 550 nm.
Plasma ic 8-Hyd oxy-2-Deoxyguanosine
(8-OHdG) Quan i ica ion
The plasma ic le els o 8-OHdG we e measu ed using
a compe i i e quan i a i e ELISA Ki (8-hyd oxy-2-
deoxyguanosine ELISA Ki , Abcam), acco ding o manu ac u e
ins uc ions, in a Syne gyTM mul i-mode mic opla e eade
(Gonçal es e al., 2017).
NFE2L2 and KEAP1 Genes Exp ession
Analysis
To al RNA was isola ed om pe iphe al blood samples ob ained
om MDS pa ien s using he Quick-RNATM MiniP ep (Zymo
Resea ch), acco ding o he manu ac u e ’s ins uc ions. Real-
ime quan i a i e PCR (qPCR) s udies we e pe o med o
quan i y he NFE2L2 and KEAP1 genes (no malized o HPRT
gene). A e ex ac ion, o al RNA was e e se ansc ibed
in o cDNA wi h Supe Sc ip TM III Re e se T ansc ip ase ki
(In i ogen, Li e Technologies) using a 1:1 mix o andom
F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g 3June 2021 | Volume 9 | A icle 701328
cell-09-701328 June 4, 2021 Time: 12:12 # 4
Gonçal es e al. Oxida i e S ess as ESA-Response Bioma ke
hexame s and oligo-dTs. Then, NFE2L2, KEAP1, and HPRT
genes we e ampli ied in duplica e using SsoFas TM E aG eenR

Supe mixe (BioRad) in an IQ5 Real-Time PCR Sys em (BioRad).
To assess he eac ion e iciency, s anda d cu es we e c ea ed
o all s udied genes using a se ially dilu ed con ol sample. Fo
each expe imen was included a no empla e con ol (NTC) as he
nega i e con ol. The speci ici y o qPCR eac ions was con i med
using he mel ing cu e analysis. The ela i e exp ession o he
a ge genes was analyzed using he 21C o mula.
S a is ical Analysis
S a is ical analysis was pe o med using SPSS e sion 26.0, and
g aphics we e cons uc ed h ough G aphPad P ism e sion 6.0.
Con inuous a iables we e exp essed as mean ±SEM (s anda d
e o o he mean), unless o he wise speci ied, and ca ego ical
a iables as numbe s and pe cen ages. To accoun o changes in
plasma lipid con en , i amin A and E we e exp essed in ela ion
o choles e ol because lipids a ec he concen a ion o hese
i amins. All s a is ical analyses we e wo-sided, and a p<0.05
was conside ed s a is ically signi ican . No mali y was assessed
by he Kolmogo o -Smi no es . Fo no mally dis ibu ed
con inuous a iables, he S uden ’s - es was pe o med o assess
he s a is ical signi icance o he di e ence be ween means o
ESA esponde s and non- esponde s. When con inuous a iables
did no show no mal dis ibu ion, he Mann-Whi ney U was
used. Logis ic eg ession, adjus ed o age and gende , was
pe o med o es ablish he ac o s ha we e associa ed wi h
ESA esponse. Fac o s ha showed a signi ican associa ion
in he uni a ia e analysis we e included in he mul i a ia e
logis ic eg ession o de e mine he associa ed independen
a iables. The calib a ion o logis ic models was assessed by he
Hosme -Lemeshow goodness-o - he- i es . Resul s om logis ic
analysis we e exp essed as adjus ed odds a ios (OR) wi h he
co esponding 95% con idence in e al (CI). In his analysis,
he OR co esponds o a 1-uni inc ease in he explana o y
a iable. Finally, ecei e ope a ing cha ac e is ic (ROC) cu es
we e pe o med o e alua e he accu acy o signi ican pa ame e s
as ESA he apy esponse bioma ke . The a ea unde he cu e
(AUC) was calcula ed as a measu emen o he accu acy o he
es , and an op imal cu -o poin was de e mined as he alue
o he pa ame e ha maximized he sum o speci ici y and
sensi i i y (Youden’s J Index).
RESULTS
Biodemog aphic and Clinical
Cha ac e is ics o MDS Pa ien s
The p esen s udy en olled 66 pa ien s diagnosed wi h MDS
[median age o 74 yea s ( ange 22–89), 60.1% (n= 40) emales
and 39.9% (n= 26) males]. Table 1 show he biodemog aphical
and clinical cha ac e is ics o MDS pa icipan s. MDS pa ien s
we e diagnosed acco ding o WHO classi ica ion (2016). Nine
(13.6%) we e diagnosed wi h MDS-SLD, 10 (15.2%) wi h MDS-
RS, 40 (60.6%) wi h MDS-MD, and se en (10.6%) wi h MDS-EB.
The IPSS-R p ognos ic sco e was lowe in 38 (57.6%) pa ien s,
highe in 13 (19.7%), and no epo ed in 15 (22.7%). In i een
MDS pa ien s cy ogene ic abno mali ies we e de ec ed, ha ing
37 pa ien s good cy ogene ic, 11 in e media e, and wo poo .
MDS pa ien s had been ans used wi h a median o 24 U/l
o sEPO, anging om 4 o 494 U/l. F om hese pa ien s,
44 (66.7%) ecei ed ESA- ea men as suppo i e ca e, and 20
(45.5%) did no espond o ea men . All pa ien s ha ecei ed
ESA- ea men had sEPO le els below 500 U/l (median o 35 U/l,
anging om 4 o 494 U/l).
Oxida i e S ess Le els in ESA-T ea ed
Pa ien s
To in es iga e he in ol emen o oxida i e s ess in he
esponse o ESA- ea men , he plasma ic le els o eac i e
oxygen/ni ogen species (pe oxides and NO), non-enzyma ic
an ioxidan de enses [u ic acid, i amin E (plasma ic and
e y h ocy ic), i amin A, GSH, GSSG, TAS], enzyma ic
de enses (e y h ocy e GPX and GR ac i i ies), and he le els
o mac omolecules oxida i e damage [8-OH-dG and MDA
(plasma ic and e y h ocy ic)] we e compa ed be ween pa ien s
ha esponded and did no espond o ESA ea men (Figu e 1
and Table 2). The pe oxides/TAS and NO/TAS a ios we e
calcula ed o analyze he oxida i e s ess s a us o MDS pa ien s.
Mo eo e , we analyzed he exp ession o NFE2L2 gene, ha
encode he ansc ip ion ac o NRF2 (a edox egula o ), and i s
nega i e egula o , he KEAP1 gene.
As shown in Figu e 1, pa ien s ha espond o ESA ea men
showed lowe le els o plasma pe oxides (3.48 ±0.21 µM;
p<0.001), cellula GSH [median (Med): 5.93, in e qua ile ange
(IqR): 1.39 µmol/g Hb; p<0.001], and cellula GR ac i i y
(Med: 5.89, IqR: 3.39 U/g Hb; p= 0.001) in compa ison o
pa ien s who did no espond o ESA ea men (pe oxides:
5.65 ±0.21 µM; GSH: Med: 7.99, IqR: 7.39 U/g Hb; GR:
Med: 9.06, IqR: 4.83 U/g Hb). ESA esponde s also showed
lowe le els o pe oxides/TAS a io (3.60 ±0.24, p<0.001)
compa ed o hose wi hou esponse (6.17 ±0.38 µM). Mo eo e ,
MDS pa ien s ha espond o ESA ea men showed highe
exp ession le els o he NFE2L2 gene (Med: 4.544, IqR: 6.150;
p= 0.001) han hose ha did no espond o ESA ea men
(Med: 1.945, IqR: 2.590; Figu e 2). No di e ences we e obse ed
be ween MDS sub ypes.
Oxida i e S ess Le els as P edic i e
Bioma ke o ESA T ea men Response
The associa ion be ween oxida i e s ess pa ame e s and ESA
esponse was analyzed h ough logis ic eg ession (Table 3).
The le els o plasma ic pe oxides, he pe oxides/TAS a io, he
cellula GSH and GR ac i i y we e signi ican ly associa ed wi h
ESA ea men in he uni a ia e analysis. In he mul i a ia e
analysis, pe oxides we e signi ican ly associa ed wi h ESA non-
esponse [Odds a io (OR): 0.111; 95% con idence in e al (CI):
sensi i i y: 92%; speci ici y: 90%; posi i e p edic i e alue (PPV):
96%; nega i e p edic i e alue (NPV): 80%; p= 0.007]. We did
no ind any associa ion be ween sEPO le els and he esponse
o ESA ea men .
The po en ial o oxida i e s ess pa ame e s as p edic i e
bioma ke s o esponse o ESA ea men was assessed by
F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g 4June 2021 | Volume 9 | A icle 701328
cell-09-701328 June 4, 2021 Time: 12:12 # 5
Gonçal es e al. Oxida i e S ess as ESA-Response Bioma ke
TABLE 1 | Biodemog aphic and clinical cha ac e is ics o MDS pa ien s.
Cha ac e is ics MDS (n= 66) ESA- ea ed MDS (n= 44)
Demog aphic ea u es
Gende (%)
Male 26 (39.9) 18 (40.9)
Female 40 (60.1) 26 (59.1)
Age (yea s)
Median 74 79
Range 22–89 47–87
Clinical ea u es
Hema ological pa ame e s (median, ange)
WBC (×109/l) 3.5 (1.3–13.0) 4.2 (1.3–13.0)
Hb (g/l) 10.6 (5.4–16.0) 9.9 (5.4–12.3)
Pla ele s (×109/l) 98 (12–324) 107 (12–317)
Se um e y h opoie in (U/l; median, ange) 24 (4–494) 35 (4–494)
Se um e i in (ng/ml; median, ange) 237 (17–1750) 191 (28–1750)
Vi amin B12 (pg/ml; median, ange) 608 (236–2000) 799 (236–2000)
Folic acid (ng/ml; median, ange) 9.7 (2.2–24.0) 10.3 (4.0–24.0)
WHO 2016 classi ica ion
MDS-SLD (%) 9 (13.6) 4 (9.1)
MDS-RS (%) 10 (15.2) 10 (22.7)
MDS-MD (%) 40 (60.6) 30 (68.2)
MDS-EB (%) 7 (10.6) 0 (0)
IPSS-R isk g oups
Lowe - isk (IPSS-R sco e ≤3.5) 38 32
Highe - isk (IPSS-R sco e >3.5) 13 0
No eco ded 15 12
Cy ogene ics
Good 37 22
In e media e 11 10
Poo 2 0
No eco ded 15 12
MDS, myelodysplas ic synd ome; ESA, e y h opoie in-s imula ing agen s; WHO, Wo ld Heal h O ganiza ion; WBC, whi e blood cells; Hb, hemoglobin; MDS-SLD, MDS
wi h single lineage dysplasia; MDS-MD, MDS wi h mul ilineage dysplasia; MDS-RS, MDS wi h ing side oblas s; MDS-EB, MDS wi h excess blas s; IPSS-R, in e na ional
p ognos ic sco ing sys em e ised.
ROC cu es (Table 4 and Figu e 3). The plasma ic pe oxides
le els and he pe oxides/TAS a io we e he mos accu a e
bioma ke o ESA esponse, wi h an a ea unde he cu e
(AUC) o 0.946 [95% CI: 0.883–1.000; p<0.001] and 0.918
(95% CI: 0.838–1.000; p<0.001), espec i ely. The pe oxides
le els highe han 4.96 µM (sensi i i y: 80%; speci ici y: 100%;
PPV: 100%; NPV: 86%) and he pe oxides/TAS a io highe
han 3.94 (sensi i i y: 100%; speci ici y: 75%; PPV: 77%; NPV:
100%) we e de ined as he op imal cu -o alues o iden i y
ESA non- esponde MDS pa ien s. The GR ac i i y, he GSH
le els and he NFE2L2 gene exp ession also showed po en ial
as ESA esponse bioma ke s. Despi e i s lowe po en ial, he
GSH e y h ocy es le els (AUC = 0.814; 95% IC: 0.690–0.937;
p<0.001) show o be also good bioma ke s o ESA esponse.
The bes GSH cu -o alues we e 8.86 µmol/g Hb (sensi i i y:
45%; speci ici y: 100%; PPV: 100%; NPV: 69%). The GR ac i i y
(AUC: 0.793; 95% CI: 0.661–0.924; p= 0.001; sensi i i y:
90%; speci ici y: 58%; PPV: 64%; NPV: 88%) and he NFE2L2
gene exp ession le els (AUC: 0.785; 95% CI: 0.653–0.918;
p= 0.001; sensi i i y: 90%; speci ici y: 55%; PPV: 62%; NPV:
87%) we e conside ed he bioma ke s wi h lowe po en ial o
p edic ESA esponse.
DISCUSSION
The cu en s udy aimed o unde s and be e he mechanisms
o esis ance o ESA he apy and he in luence o oxida i e
s ess in ea men esponse in lowe - isk MDS pa ien s. We
ound ha pa ien s who espond o ESA ea men ha e
lowe le els o plasma pe oxides, cellula GSH, cellula GR
ac i i y, pe oxides/TAS a io, and highe le els o NFE2L2 gene
exp ession in compa ison o pa ien s who did no espond
o ESA ea men . These pa ame e s a e good bioma ke s o
he apy esponse, wi h pe oxides le els being he bes and
accu a e bioma ke o ESA esponse. To he bes o ou
knowledge, only one s udy analyzed he associa ion be ween
oxida i e s ess and ESA esponse. In his s udy, Khalil e al.
(2016) ound ha pa ien s wi h an end-s age enal disease wi h
lowe e y h ocy e supe oxide dismu ase and highe MDA le els
F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g 5June 2021 | Volume 9 | A icle 701328

cell-09-701328 June 4, 2021 Time: 12:12 # 6
Gonçal es e al. Oxida i e S ess as ESA-Response Bioma ke
FIGURE 1 | Analysis o oxida i e s ess pa ame e s in pa ien s wi h myelodysplas ic synd ome acco ding o hei esponse o ESA ea men . The ollowing oxida i e
s ess le els a e ep esen ed: (A) plasma ic pe oxides, (B) e y h ocy e educed glu a hione (GSH), (C) e y h ocy e glu a hione educ ase (GR), and (D) pe oxides/ o al
an ioxidan s a us (TAS) a io. ***p≤0.001.
TABLE 2 | Analysis o oxida i e s ess pa ame e s in pa ien s wi h myelodysplas ic synd ome acco ding o ESA esponse ea men .
Pa ame e ESA esponde s ESA non- esponde s p- alue
Mean SEM Mean SEM
U ic acid (mg/dl) 0.40 0.03 0.40 0.04 0.944
pVi amin A (µM/mM) 0.45 0.04 0.43 0.03 0.962
pVi amin E (µM/mM) 6.65 0.31 6.02 0.30 0.203
eVi amin E (nmol/gHb)* 23.5 16.8 46.6 60.4 0.059
pMDA (µM) 0.84 0.05 0.75 0.05 0.190
eMDA (µmol/g Hb) 81.6 8.5 53.3 4.8 0.268
NO (µM) 11.8 1.2 13.1 1.5 0.450
eGPx (U/g Hb)* 4.50 4.42 4.85 6.46 0.409
8-OH-dG (ng/ml) 34.2 1.4 39.7 1.0 0.069
TAS (mM)* 1.04 0.29 0.96 0.12 0.257
NO/TAS a io* 14.4 14.0 13.3 7.9 0.723
*These alues a e ep esen ed as median and in e qua ile ange. Plasma ic i amin A and E alues a e ep esen ed as i amin A:choles e ol and i amin E:choles e ol
a ios, espec i ely. p, plasma ic; e, e y h ocy ic; 8-OH-dG, 8-hyd oxy-20-deoxyguanosine; NO, ni ic oxide; GSH, educed glu a hione; TAS, o al an ioxidan s a us; MDA,
malondialdehyde; SEM, s anda d e o o he mean.
show poo esponse o ESA ea men (Khalil e al., 2016).
He e we ound ha esponse o ESA ea men is associa ed
wi h a dec ease in pe oxides le els and pe oxides/TAS a io
and a dec ease in GSH le els and GR ac i i y. Al hough we
obse ed an inc ease in hese an ioxidan de enses on non-
esponde s. Globally hese pa ien s p esen ed a oxida i e s ess
F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g 6June 2021 | Volume 9 | A icle 701328
cell-09-701328 June 4, 2021 Time: 12:12 # 7
Gonçal es e al. Oxida i e S ess as ESA-Response Bioma ke
FIGURE 2 | Exp ession o NFE2L2 and KEAP1 genes in pa ien s wi h myelodysplas ic synd ome acco ding o hei esponse o ESA ea men . The RNA exp ession
o NFE2L2 and KEAP1 genes we e no malized o he HPRT gene (endogenous con ol). ***p≤0.001.
TABLE 3 | Signi ican oxida i e s ess pa ame e s as ESA esponse bioma ke in myelodysplas ic synd ome.
Bioma ke s AUC Cu -o
alue (95% CI) p- alue alue SEN (%) SPE (%) PPV (%) NPV (%)
Pe oxide (µM) 0.946 (0.883–1.000) <0.001 >4.96 80 100 100 86
GR (U/g Hb) 0.793 (0.661–0.924) 0.001 >5.97 90 58 64 88
GSH (µmol/g Hb) 0.814 (0.690 –0.937) <0.001 >8.86 45 100 100 69
Pe oxides/TAS a io 0.918 (0.838–1.000) <0.001 >3.94 100 75 77 100
NFE2L2 gene exp ession 0.785 (0.653–0.918) 0.001 <4.23 90 54 62 87
OR, odds a io; CI, con idence in e al; GR, glu a hione edu ase; GSH, educed glu a hione; TAS, o al an ioxidan s a us; AUC, a ea unde he cu e; CI, con idence
in e al; SEN, sensi i i y; SPE, speci ici y; PPV, posi i e p edic i e alue; NPV, nega i e p edic i e alue.
TABLE 4 | P edic i e po en ial o oxida i e s ess pa ame e s as bioma ke s o ESA esponse.
Bioma ke s Uni a ia e Mul i a ia e
OR (95% CI) p- alue OR (95% CI) p- alue
Pe oxide (µM) 0.088 (0.021–0.371) 0.001 0.137 (0.024–0.787) 0.026
GR (U/g Hb) 0.519 (0.315–0.855) 0.010 0.207 (0.018–2.447) 0.211
GSH (µmol/g Hb) 0.644 (0.477–0.870) 0.004 0.631 (0.310–1.283) 0.203
Pe oxide/TAS a io§0.252 (0.117–0.541) 0.001 – –
NFE2L2 gene exp ession 1.707 (1.177–2.477) 0.005 3.555 (0.848–14.900) 0.830
§Due o mul icollinea i y be ween pe oxide and pe oxide/TAS a io, he las one was no included in mul i a ia e analysis. OR, odds a io; CI, con idence in e al; GR,
glu a hione edu ase; GSH, educed glu a hione; TAS, o al an ioxidan s a us.
s a e ha is in acco dance wi h o he s udies ha show ha
oxida i e s ess is associa ed wi h d ug esis ance in cance (Pou
Kha a i e al., 2018;Mau ya e al., 2021). ROS is known o
i s abili y o induce mu a ions and p omo e cance cell g ow h
and an icance d ug esis ance. The oxida i e s ess in cance
cells has been shown o co ela e wi h he agg essi eness o
umo s and poo su i al o pa ien s wi h cance (T achoo ham
e al., 2009). Oxida i e s ess is excep ionally impo an o
e y h opoiesis and is in ol ed in p oli e a ion, su i al, and
di e en ia ion o e y h oid cell p ogeni o s in esponse o EPO
binding o e y h opoie in ecep o , and can also induce cell
dea h by apop osis (Gha a i, 2008). Since oxida i e s ess has
dele e ious e ec s on e y h oid p ogeni o cells, he inc ease in
oxida i e s ess a io and pe oxides could induce e y h ocy es
apop osis/ine icien e y h opoiesis and, he e o e, jus i y he
non- esponde pheno ype obse ed in his s udy. Addi ionally,
he ansc ip ion ac o NRF2 is conside ed a p ognos ic
bioma ke in cance , wi h e y high le els associa ed wi h poo
esponse o an icance d ugs (F ijho e al., 2015). Howe e ,
we ound ha MDS pa ien s ha did no espond o ESA
ea men ha e lowe le els o NFE2L2, he gene ha encode
he NRF2 ansc ip ion ac o . This esul is in ag eemen
wi h he highe le els o pe oxides and oxida i e s ess a io
obse ed in hese pa ien s. Fu he mo e, we p e iously ound
F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g 7June 2021 | Volume 9 | A icle 701328
cell-09-701328 June 4, 2021 Time: 12:12 # 8
Gonçal es e al. Oxida i e S ess as ESA-Response Bioma ke
FIGURE 3 | The p edic i e alue o pe oxides and pe oxides/TAS a io
ega ding ESA ea men esponses in MDS pa ien s. A ROC cu e analysis
o pe oxides and pe oxides/TAS a io was pe o med o assess i s po en ial
as a bioma ke o ESA ea men esponse.
ha oxida i e s ess pa ame e s and he 1ψmi a e diagnos ic
bioma ke s and su i al p edic o s o MDS, wi h GSH le els
p o iding he mos accu a e and eliable indica o o MDS
diagnosis and su i al (Gonçal es e al., 2015). Addi ionally,
in ano he s udy we ound ha GSH le els co ela e wi h he
elapse and su i al o acu e lymphoblas ic leukemia pa ien s
(Sa men o-Ribei o e al., 2012).
Se e al s udies ha e examined ac o s, in lowe - isk MDS
pa ien s, ha could be used o p edic esponse o ESA ea men
and o shape ea men s mo e e icien ly. One o he mos
s udied ac o s in esponse o ESAs associa ed wi h o wi hou
G-CSF is he sEPO le els, and nume ous s udies ha e epo ed
co ela ions be ween sEPO and he apy esponse. The majo i y
o hese s udies used a sEPO cu o o 100 U/L, wi h esponse
a es anging om 50 o 93% o pa ien s wi h sEPO <100
U/L e sus 12–58% o pa ien s wi h sEPO >100 U/L (Pa k
e al., 2020). In he p esen s udy we did no ind any associa ion
be ween sEPO and ESA esponse, bu he esponse a es we e
58% o pa ien s wi h sEPO <100 U/L and 38% o pa ien s
wi h sEPO >100 U/L. Howe e , in a p e ious s udy including
102 MDS pa ien s, we ound ha sEPO is a p edic i e ac o
o esponse o he apy wi h subcu aneous EPO (Co esão e al.,
2015). Se e al o he ac o s ha e also been s udied including
absolu e neu ophil coun (S asi e al., 2005), cy openia le els
(Mol eni e al., 2013), hemoglobin le els (S asi e al., 2005;
San ini e al., 2013;Hous on e al., 2017), pla ele coun (S asi
e al., 2005;San ini e al., 2013), age (S asi e al., 2005;Hous on
e al., 2017), gende (S asi e al., 2005;Hous on e al., 2017),
bu s - o ming uni -e y h oid le els (F isan e al., 2010), bone
ma ow blas s (S asi e al., 2005;F isan e al., 2010;San ini e al.,
2013), IPSS s a us (S asi e al., 2005;F isan e al., 2010;San ini
e al., 2013), p-ERK1/2 le els (F isan e al., 2010), se um TNF-
alpha (S asi e al., 2005), soma ic mu a ions (Kosmide e al.,
2016), among o he s.
This wo k has some limi a ions ha mus be aken in o
accoun . We ec ui ed almos all pa ien s newly diagnosed
wi h MDS du ing ec ui men ime, bu we we e only able
o s udy a ela i ely small coho o pa ien s, especially
hose ea ed wi h ESA. The oxida i e s ess pa ame e s we e
only analyzed in pe iphe al blood samples (plasma, o al
leucocy es, and/o e y h ocy es). Al hough MDS is a clonal
s em cell diso de , he same s udies mus be epea ed in
e y h oid p ecu so cells ob ained om bone ma ow samples.
Howe e , his ac may also be one o he wo k s eng hs,
since pe iphe al blood is a mo e accessible and less in asi e
biological sample.
In conclusion, he p esen epo demons a ed a co ela ion
be ween oxida i e s ess le els and he esponse o ESA ea men
in lowe - isk MDS pa ien s. We ound ha pe oxides le els and
pe oxides/TAS a io a e good and accu a e pe iphe al bioma ke s
ha p edic pa ien s ha will no espond o ESA he apy.
DATA AVAILABILITY STATEMENT
The aw da a suppo ing he conclusions o his a icle will be
made a ailable on eques o he co esponding au ho .
ETHICS STATEMENT
The s udies in ol ing human pa icipan s we e e iewed and
app o ed by E hics Commi ee o he Facul y o Medicine
o he Uni e si y o Coimb a Azinhaga de San a Comba,
Coimb a, Po ugal. The pa ien s/pa icipan s p o ided hei
w i en in o med consen o pa icipa e in his s udy.
AUTHOR CONTRIBUTIONS
AG and AS-R concei ed he s udy. AG, RA, IB, AP, and JJ
pe o med he expe imen s. EC and BM ec ui ed and collec ed
pa ien da a. AG and BO analyzed and in e p e ed he da a. AG
d a ed he manusc ip . AS-R and JN e iewed and edi ed he
manusc ip . All au ho s ead and app o ed he inal manusc ip .
FUNDING
The p esen wo k was suppo ed by CIMAGO –
Cen e o In es iga ion on En i onmen , Gene ics and
Oncobiology, Facul y o Medicine, Uni e si y o Coimb a,
Po ugal and by Na ional Funds ia FCT (Founda ion
o Science and Technology) h ough he S a egic P ojec
UID/NEU/04539/2019, COMPETE-FEDER (POCI-01-0145-
FEDER-007440), UIDB/04539/2020, and UIDP/04539/2020
(CIBB). FCT suppo ed JJ wi h a g an (SFRH/BD/145531/2019).
F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g 8June 2021 | Volume 9 | A icle 701328
cell-09-701328 June 4, 2021 Time: 12:12 # 9
Gonçal es e al. Oxida i e S ess as ESA-Response Bioma ke
REFERENCES
Adès, L., I zykson, R., and Fenaux, P. (2014). Myelodysplas ic synd omes. Lance .
383, 2239–2252.
A be , D. A., O azi, A., Hasse jian, R., Thiele, J., Bo owi z, M. J., Le Beau, M. M.,
e al. (2016). The 2016 e ision o he Wo ld Heal h O ganiza ion classi ica ion
o myeloid neoplasms and acu e leukemia. Blood 127, 2391–2405. doi: 10.1182/
blood-2016-03-643544
Baldei as, I., San ana, I., P oença, M. T., Ga ucho, M. H., Pascoal, R., Rod igues,
A., e al. (2010). Oxida i e damage and p og ession o Alzheime ’s disease in
pa ien s wi h mild cogni i e impai men . J. Alzheime s Dis. 21, 1165–1177.
Ba ham, D., and T inde , P. (1972). An imp o ed colou eagen o he
de e mina ion o blood glucose by he oxidase sys em. Analys 97, 142–145.
doi: 10.1039/an9729700142
Beneduce, E., Ma e, A., De Falco, L., Mbiandjeu, S., Chiab ando, D., Tolosano, E.,
e al. (2019). Fyn kinase is a no el modula o o e y h opoie in signaling and
s ess e y h opoiesis. Am. J. Hema ol. 94, 10–20. doi: 10.1002/ajh.25295
Cazzola, M. (2020). Myelodysplas ic Synd omes. N. Engl. J. Med. 383, 1358–1374.
Co esão, E., Ten ei o, R., Ramos, S., Pe ei a, M., Césa , P., Ca da, J. P.,
e al. (2015). E i opoie ina Sé ica como Ma cado P ognós ico em
Sínd ome Mielodisplásica [Se um E y h opoie in as P ognos ic Ma ke in
Myelodysplas ic Synd omes]. Ac a Med. Po . 28, 720–725.
De Leenhee , A. P., De Be e e, V. O., and Claeys, A. E. (1979). Measu emen o
alpha-, be a-, and gamma ocophe ol in se um by liquid ch oma og aphy. Clin.
Chem. 25, 425–428. doi: 10.1093/clinchem/25.3.425
F ijho , J., Winya d, P. G., Za ko ic, N., Da ies, S. S., S ocke , R., Cheng, D., e al.
(2015). Clinical Rele ance o Bioma ke s o Oxida i e S ess. An ioxid. Redox
Signal. 23, 1144–1170.
F isan, E., Pawlikowska, P., Pie e-Eugene, C., Viallon, V., Gibaul , L., Pa k, S., e al.
(2010). p-ERK1/2 is a p edic i e ac o o esponse o e y h opoiesis s imula ing
agen s in low/in -1 myelodysplas ic synd omes. Haema ologica 95, 1964–1968.
doi: 10.3324/haema ol.2010.024349
Ga cia-Mane o, G. (2010). P ognosis o myelodysplas ic synd omes. Hema ology
Am. Soc. Hema ol. Educ. P og am 2010, 330–337. doi: 10.1182/asheduca ion-
2010.1.330
Gha a i, S. (2008). Oxida i e s ess in he egula ion o no mal and neoplas ic
hema opoiesis. An ioxid. Redox Signal. 10, 1923–1940. doi: 10.1089/a s.2008.
2142
Gho i, H., Ame , J., Winde , A., Rachmilewi z, E., and Fibach, E. (2007). Oxida i e
s ess in ed blood cells, pla ele s and polymo phonuclea leukocy es om
pa ien s wi h myelodysplas ic synd ome. Eu . J. Haema ol. 79, 463–467. doi:
10.1111/j.1600-0609.2007.00972.x
Goldbe g, D., and Spoone , R. (1983). “Glu a hione educ ase,” in Me hods o
enzyma ic analysis 3 d ed, eds H. Be gmeye , J. Be gmeye , and M. G assl
(Weinheim: Ve lag Chemie), 258–265.
Gonçal es, A. C., Al es, R., Baldei as, I., Co esão, E., Ca da, J. P., B anco,
C. C., e al. (2017). Gene ic a ian s in ol ed in oxida i e s ess, base excision
epai , DNA me hyla ion, and ola e me abolism pa hways in luence myeloid
neoplasias suscep ibili y and p ognosis. Mol. Ca cinog. 56, 130–148. doi: 10.
1002/mc.22478
Gonçal es, A. C., Co esão, E., Oli ei os, B., Al es, V., Espadana, A. I., Ri o,
L., e al. (2015). Oxida i e s ess and mi ochond ial dys unc ion play a
ole in myelodysplas ic synd ome de elopmen , diagnosis, and p ognosis: a
pilo s udy. F ee Radic Res. 49, 1081–1094. doi: 10.3109/10715762.2015.103
5268
Hasselbalch, H. C., Thomassen, M., Hasselbalch Riley, C., Kj´
c , L., S au e La sen,
T., Jensen, M. K., e al. (2014). Whole Blood T ansc ip ional P o iling Re eals
De egula ion o Oxida i e and An ioxida i e De ence Genes in Myelo ib osis
and Rela ed Neoplasms. Po en ial Implica ions o Down egula ion o N 2
o Genomic Ins abili y and Disease P og ession. PLoS One 9:e112786. doi:
10.1371/jou nal.pone.0112786
Hous on, B. L., Jayaka , J., Wells, R. A., Lenis, M., Zhang, L., Zhu,
N., e al. (2017). A p edic i e model o esponse o e y h opoie in
s imula ing agen s in myelodysplas ic synd ome: om he Canadian MDS
pa ien egis y. Ann. Hema ol. 96, 2025–2029. doi: 10.1007/s00277-017-
3137-0
Imbesi, S., Musolino, C., Alleg a, A., Saija, A., Mo abi o, F., Calapai, G., e al.
(2013). Oxida i e s ess in oncohema ologic diseases: an upda e. Expe Re .
Hema ol. 6, 317–325. doi: 10.1586/ehm.13.21
Jhanwa , S. C. (2015). Gene ic and epigene ic pa hways in myelodysplas ic
synd omes: a b ie o e iew. Ad . Biol. Regul. 58, 28–37. doi: 10.1016/j.jbio .
2014.11.002
Khalil, S. K., Ame , H. A., El Behai y, A. M., and Wa da, M. (2016). Oxida i e s ess
du ing e y h opoie in hypo esponsi eness anemia a end s age enal disease:
molecula and biochemical s udies. J. Ad . Res. 7, 348–358. doi: 10.1016/j.ja e.
2016.02.004
Kom okji, R. S., Seke es, M. A., and Lis , A. F. (2011). Managemen o lowe - isk
myelodysplas ic synd omes: he a and e idence. Cu . Hema ol. Malig. Rep. 6,
145–153. doi: 10.1007/s11899-011-0086-x
Kosmide , O., Passe , M., San ini, V., Pla zbecke , U., And ieu, V., Zini, G.,
e al. (2016). A e soma ic mu a ions p edic i e o esponse o e y h opoiesis
s imula ing agen s in lowe isk myelodysplas ic synd omes? Haema ologica
2016, e280–e283.
Lin, P., Ren, Y., Yan, X., Luo, Y., Zhang, H., Kesa wani, M., e al. (2019). The
high NRF2 exp ession con e s chemo he apy esis ance pa ly h ough up-
egula ed DUSP1 in myelodysplas ic synd omes. Haema ologica 104, 485–496.
doi: 10.3324/haema ol.2018.197749
Liu, Y., Li, Q., Zhou, L., Xie, N., Nice, E. C., Zhang, H., e al. (2016). Cance
d ug esis ance: edox ese ing ende s a way. Onco a ge 7, 42740–42761.
doi: 10.18632/onco a ge .8600
Mau ya, R. P., P ajapa , M. K., Singh, V. P., Roy, M., Todi, R., Bosak,
S., e al. (2021). Se um Malondialdehyde as a Bioma ke o Oxida i e
S ess in Pa ien s wi h P ima y Ocula Ca cinoma: impac on Response
o Chemo he apy. Clin. Oph halmol. 15, 871–879. doi: 10.2147/op h.s28
7747
Mol eni, A., Ri a, M., G eco, R., Nichela i, M., Ra ano, E., Ma bello, L.,
e al. (2013). Ve i ying Hells om-Lindbe g sco e as p edic i e ool o
esponse o e y h opoie in he apy acco ding o he “In e na ional Wo king
G oup” c i e ia, in anemic pa ien s a ec ed by myelodysplas ic synd ome: a
monocen ic expe ience. In . J. Hema ol. 97, 472–479. doi: 10.1007/s12185-
013-1305-0
Mu i, G. J. (2004). Pa hobiology, classi ica ion, and diagnosis o myelodysplas ic
synd ome. Res. Clin. Haema ol. 17, 543–557. doi: 10.1016/s1521-6926(04)
00073-8
Paglia, D. E., and Valen ine, W. N. (1967). S udies on he quan i a i e and
quali a i e cha ac e iza ion o e y h ocy e glu a hione pe oxidase. J. Lab. Clin.
Med. 70, 158–169.
Pa k, S., Kelaidi, C., Meunie , M., Casade all, N., Ge ds, A. T., and Pla zbecke ,
U. (2020). The p ognos ic alue o se um e y h opoie in in pa ien s
wi h lowe - isk myelodysplas ic synd omes: a e iew o he li e a u e and
expe opinion. Ann. Hema ol. 99, 7–19. doi: 10.1007/s00277-019-03
799-4
Pou Kha a i, A., Liu, Y., He, E., Skog, S., and Haghdoos , S. (2018). Se um
8-Oxo-dG as a P edic o o Sensi i i y and Ou come o Radio he apy and
Chemo he apy o Uppe Gas oin es inal Tumou s. Oxid. Med. Cell. Longe .
2018:4153574. doi: 10.1155/2018/4153574
San ini, V., Schemenau, J., Le is, A., Ballea i, E., Sapena, R., Ades, L., e al. (2013).
Can he e ised IPSS p edic esponse o e y h opoie ic-s imula ing agen s in
pa ien s wi h classical IPSS low o in e media e-1 MDS? Blood 122, 2286–2288.
doi: 10.1182/blood-2013-07-512442
Sa dina, J. L., López-Ruano, G., Sánchez-Sánchez, B., Llanillo, M., and He nández-
He nández, A. (2012). Reac i e oxygen species: a e hey impo an o
haema opoiesis?. C i . Re . Oncol. Hema ol. 81, 257–274. doi: 10.1016/j.
c i e onc.2011.03.005
Sa men o-Ribei o, A. B., P oença, M. T., Sousa, I., Pe ei a, A., Guedes, F., Teixei a,
A., e al. (2012). A possible ole o oxida ion s ess in lymphoid leukaemias
and he apeu ic ailu e. Leuk. Res. 36, 1041–1048. doi: 10.1016/j.leuk es.2012.
03.015
S asi, R., Ab uzzese, E., Lanze a, G., Te zoli, E., and Amado i, S. (2005).
Da bepoe in al a o he ea men o anemic pa ien s wi h low and
in e media e-1- isk myelodysplas ic synd omes. Ann. Oncol. 16, 1921–1927.
doi: 10.1093/annonc/mdi400
F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g 9June 2021 | Volume 9 | A icle 701328