Oxidative Stress Parameters Can Predict the Response to Erythropoiesis-Stimulating Agents in Myelodysplastic Syndrome Patients

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). 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