Telomere length and human telomerase reverse transcriptase (hTERT) level in patients with acute myeloid leukemia: Impact on clinical outcome

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Original research article/Praca oryginalna

Telomere length and human telomerase reverse transcriptase (hTERT) level in patients with acute myeloid leukemia: Impact on clinical outcome

Samia F. El Belbesy, Hanaa A. El Aggan, Hala K. Sultan, Amel A. El Naggar *, Hayat K. Fadlalla Ahmed

HaematologyDepartment,MedicalResearchInstitute,AlexandriaUniversity,Egypt

article info

Articlehistory:

Received:02.12.2014 Accepted:15.07.2015 Availableonline:26.07.2015

Keywords:

Acutemyeloidleukemia

hTERT

Telomerelength

Responsetotherapy

abstract

Theresponsetotreatment andoverall survivalofpatientswithAMLisheterogeneous.

Prognostic factorsare urgently needed in order to beable to better predict treatment outcomes.Theaimofthepresentworkwastostudytelomerelengthandhumantelo- merase reversetranscriptase (hTERT)level inacute myeloid leukemiaand todetectif theseparametersmightbeusefulinprovidinginsightintotheclinicaloutcomeofAML patients.ELISAtechniquewasusedtomeasurehTERTlevelquantitativePCRformeasu- ringtelomerelength.Thestudyincluded70individuals,50patientswithacutemyeloid leukemiaandtwentyhealthyindividualswithcomparableageandsex.Therewasstatis- tically significant higher level of hTERT andlower RTLin patients thancontrols. The patients were treated according to the standard chemotherapy protocol for induction andtheywerefollowedupbybonemarrowexamination.MeanhTERTlevelinpatients whodidnotachievecompletehematologicalremission wasstatisticallysignificanthig- her than that in patients whoachieved complete hematological remission (48.87 and 34.32respectively) (z= 1.98,p=0.048). Mean RTL inpatients who achieved remission washigherthanthatinpatientswhodidnotachieveremission(0.56and0.37respecti- vely).However,itdidnotreachstatisticalsignificance.Mediansurvivaltimeinpatients whoachievedremission wasstatisticallysignificantlongerthanthatinthose whodid notachieveremission(26msand4msrespectively).

ItwasfoundthatbothrelativetelomerelengthandhTERTcouldbeusedforassessing clinicalbehaviorandpredictingtreatmentoutcomeinAMLpatients.

*Correspondingauthorat:AlferdousStreet,Smouha,Alexandria,Egypt.Tel.:+2001225870627.

E-mailaddress:amel_elnaggar@hotmail.com(A.A.ElNaggar).

ContentslistsavailableatScienceDirect

Acta Haematologica Polonica

journal homepage:www.elsevier.com/locate/achaem

http://dx.doi.org/10.1016/j.achaem.2015.07.003

zo.o.Allrightsreserved.

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Introduction

Acutemyelogenous leukemia(AML) isa malignantdisease ofthe bonemarrow inwhichhematopoieticprecursorsare arrested in an early stage of development. Despite enor- mous insights into the molecular mechanisms of acute myeloid leukemia (AML) pathophysiology, this disease is stillfatalinthemajorityofpatients,highlightingtheurgent need for novel biomarkers useful in AML prognosis and therapy[1,2].

Telomeresarespecializednucleoproteinstructuresatthe endsof chromosomes;their functionistoprotect chromosomes from DNA breakage and to prevent chromosome fusion.Withoutnewsynthesis,telomeresundergo progres- siveshorteningwitheachcelldivision,leadingtoreplicative senescence of cells. Shortening of telomeres can result in telomereendfusionsand increasechromosomalinstability whichisakeyinitiatingeventinnumerouscancers[3].

Telomeraseisanenzymethatextendstelomericrepeats on the ends of chromosomes. Activation of telomerase enzyme isthereforerequiredfor cellsto overcomereplica- tive senescence and to be able to divide indeﬁnitely.

Telomeraseactivityisexpressedingermcellsandispresent at low level in stem cells, but is usually absent in most somaticcells.Conversely,inimmortalcancercells,telomer- aseisreactivated,andtelomeresarenotshortened,suggest- ing thattelomere elongation mightbe anessentialstep in tumorformation[4,5].

Recently genes encoding three major components of humantelomerase(TA) havebeencloned: humantelomerase RNA component (hTR), human telomerase reverse transcriptase (hTERT), and telomerase-associated protein 1 (TAP1). TERT is a telomerase catalytic subunit that is consideredasthekeycomponentforthecontroloftelomer- aseactivity[6,7].

Telomere length (TL) is a key determinant of telomere function. Accurate techniques to measure TL in human tissueshaveprovidedagreaterunderstandingoftheroleof telomeresintheprogressiontomalignancy[8].

Targeting the hTERT catalytic subunit as anticancer therapy is theoretically tumor-speciﬁc and might be less toxic due to its speciﬁc expression in tumor and highly proliferatingcells compared to other normalcells. Various newly discovered agents represent interesting anti-hTERT candidatesforclinicaldrugdevelopment[9].

The aim of the present study was to study baseline telomere length and human telomerase reverse transcriptase(hTERT)level inacutemyeloid leukemiaand todetect ifthese parametersmightbeuseful inpredicting response totherapyinacutemyeloidleukemiapatients.

Subjects

The study included seventy individuals. Fifty newly diag- nosedpatientswithAMLand twentyageandsexmatched normal healthyindividuals as a control group. They were enrolled in the study after the consent of the Ethical CommitteeoftheMedicalResearchInstitute.

The age of AML patients ranged from twenty three to seventy one years with a mean age of 50.812.97 years.

Twenty-four (48%) patients were males and 26 (52%) were females,withmaletofemaleratioof0.9.

Patients weretreatedaccordingtothestandard conven- tionalchemotherapyprotocolofAML.Thetreatmentproto- col entailed the following drugs: Daunorubicin (60mg/m²/ day for 3 days), and continuous infusion of Cytosine- Arabinoside(100mg/m²/dayfor7days)[2].Patientsunﬁtfor receivingtheinductioncyclewereexcludedfromthestudy.

Patientswereassessedforresponsetoinductiontherapy at the time of blood count recovery by bone marrow examination.Theyweredividedintotwogroups:responders who achieved complete hematological remission after induction chemotherapy and non-responders who did not achieve complete hematological remission after induction chemotherapy. The term complete remission generally refers to morphologic complete remission, deﬁned by red bloodcelltransfusionindependence,anabsoluteneutrophil count ofmorethan 1000/mlandaplateletcountof100000/

ml or greater and the presence of less than 5% blasts in a bonemarrow aspirate samplewithmarrow spicules and with a count of at least 200 nucleated cells, absence of blasts with Auer rods, and absence of extramedullary leukemia[2].

Methods

Allsubjectsparticipatinginthisstudyweresubjectedtothe following:

Thorough history taking, thorough clinical examination, routineworkupincludingliverandkidneyfunctiontests [10], radiological work up (chest X-ray, U/S abdomen

&pelvisandECHO)

Diagnostic laboratory investigations including, complete blood picture (CBP) [11], bone marrow examination [12], andimmunophenotyping[13]

QuantitativeassessmentofhTERTbyELISA[14].

KitwaspurchasedfromGenWay

quantitativePCRformeasuringtelomerelength[15].

Genomic DNA from patients was extracted using QIAampDNABloodMiniKit(50)fromQiagen.

Relative telomere length (RTL) were determined using real-timePCR.

Results

Thepresentstudyincludedﬁftypatientswithacutemyeloid leukemia, and twenty healthyindividualswith comparable ageandsex(controlgroup).

The age of AML patients ranged from twenty three to seventy oneyears witha mean age of 50.812.97 years.

Twenty-fourpatientsweremales(48%)and26werefemales (52%), with male to female ratio of 0.9. AML patients included 36patientsaged<60 years (72%),and 14patients aged60years(28%).

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According to the French-American-British (FAB) classiﬁ- cationsystemusingmorphologicandcytochemical criteria.

Thirty-one patients were M2 (62%), 9 patients were M1 (18%), 6patients were M4(12%). Other types (M3, M5, M6, andM7)wererepresentedbyonlyonecaseforeach.

Laboratoryﬁndings

In AML patients, hemoglobin concentration (Hb) ranged from4g/dlto11.5g/dl, withmeanvalueof7.52g/dl. Red bloodcell count(RBC)ranged from1.7 to4(10¹²/L),mean valueof 2.50.6 (10¹²/L). Plateletcount (Plt)ranged from 5 to 186 (10⁹/L), with mean value of 34.133.4 (10⁹/L).

Totalleucocyticcount(TLC)rangedfrom0.3to340(10⁹/L), withmeanvalueof24.150.9(10⁹/L).Blast%rangedfrom 0%to95%,withmeanvalueof50.524%.

Conventionalcytogeneticanalysiswasdonefor18patients onlyinthepresent study.Thirteen patientsshowednormal karyotype.Meanwhile,monosomy7wasdetectedin3patients, trisomy21inonepatient,andt(15,17)inonepatient.

Afterinductionchemotherapy,patientswerefollowedup by bone marrow examination to determine patient's response to therapy. Complete remission was achieved in nineteen patients (38%), and partial remission in seven patients (14%). Meanwhile, nine patients wereresistant to therapy,andﬁfteenpatientsdiedduringtheﬁrst28days.

Duringfollowup,nine outofthe nineteenpatientswho achievedCRrelapsed.

Themeanageof patientswho achievedCRafter induc- tionchemotherapy(responders)was45years. Itwasstatis- ticallysigniﬁcantlowerthanthe meanageofpatientswho did not achieve CR (nonresponders) which was 54 years (t= 2.592,p=0.013).

SerumlevelofhTERTinAMLpatientsandcontrolsubjects

SerumhTERTlevelinthecontrolgrouprangedfrom2.04to 11.11ng/ml, with a mean level of 5.062.32ng/ml.

Whereas, serum level in AML patients ranged from 4.9 to 98ng/ml,withamean levelof 43.325.4ng/ml.Therewas a statistically signiﬁcant higher level of hTERT in patients thancontrols(z= 6.107,p=0.000).

The mean serum hTERT level was higher in patients aged 60 years than in patients aged <60 years (44 and 43.1ng/mlrespectively),andinfemalescomparedtomales (43.6 and 42.9ng/ml respectively). However, this difference didnotreachstatisticalsigniﬁcance.

Relativetelomerelengthbyreal-timePCRinAMLpatientsand control

Relativetelomerelength(RTL)inAML patientsrangedfrom 0.01to1.1.Meanlevelwas0.40.3.While,incontrolgroup, it ranged from 0.85 to 6.77 withmean telomere length of 3.75.RTLwasstatistically signiﬁcantlowerinpatientsthan control(z= 6.384,p=0.000).

ThemeanRTLwaslowerinpatientsaged60yearsthan inpatientsaged<60 years(0.4and0.5respectively),andin femalescomparedtomales(0.4and 0.5respectively).How- ever,thisdifferencedidnotreachstatisticalsigniﬁcance.

CorrelationbetweenhTERTandRTL

There was a negative correlation between serum level of hTERT and the relativetelomere length (p=0.011).Level of correlation was intermediate as rs level was 0.36, as illu- stratedinFig.1.

Nostatisticallysigniﬁcantrelationwasdetectedbetween different FAB subtypes and serum HTERT level or RTL (p=0.628, p=0.475 respectively). However, when FAB subtypes M4andM5weregroupedtogetherasonegroup,AML patientswithmoredifferentiatedsubtypes(M4andM5)had statistically signiﬁcant shorter telomere length than AML patientswithlessdifferentiatedsubtypes(M1,M2,andM3).

Relative telomere length was (0.149 and 0.49 respectively) (p=0.011). But no statistical signiﬁcant difference between bothgroupsasregardhTERTlevel.

SerumhTERTlevelandrelativetelomerelengthinrelationto responsetoinductionchemotherapy

AsdemonstratedinTableI,themeanserumhTERTlevelat diagnosis inpatients whodidnot achievecompleteremission (non-responders) was statistically signiﬁcant higher than that in patients who achieved complete remission Fig.1–CorrelationbetweenhTERTandRTLinAMLpatients (r=0.36,p=0.011).Interpretationofrs:Weak(0.1–0.24);

Intermediate(0.25–0.74);Strong(0.75–0.99)

TableI–ComparisonofhTERTlevelandRTLinAML patientsaccordingtoremissionstate

Items Responders Non-responders z p hTERT(ng/ml)

Mean 34.32 48.87 1.98 0.048

SD 23.6 25.16

Relativetelomerelength

Mean 0.56 0.37 1.59 0.110

SD 0.36 0.30

Z:Mann–Whitneytest.

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(responders) (48.87 and 34.32 respectively) (z= 1.98, p=0.048).

MeanRTLinpatientswhoachievedcompleteremission (responders) was higherthan that inpatients who didnot achievecompleteremission(non-responders) (0.56and0.37 respectively).There was nostatisticalsigniﬁcant difference betweenbothgroups(z= 1.59,p=0.110).

Using ROC curve, a cut off value of 57.57ng/ml for baseline serum level of hTERT at diagnosis was calculated whichdividedthepatientsintorespondersandnon-respon- dersafterreceivingtheinductionchemotherapy.Areaunder the curve (AUC)=0.603 (overall accuracy=60.3%). This means that, in AML patients with value less or equal to 57.57ng/ml, 60.3%of patientswillachievecompleteremis- sion(sensitivitywas69.2%,andspeciﬁcitywas56%).

Similarly, a cut off value of 0.5 was calculated for baseline RTL. AUC=0.556 (overall accuracy=55.6%) with sensitivity50%andspeciﬁcity56%.Thismeansthat,inAML patientswithvalueequalormorethan0.5,55.6%ofpatients will achieve complete remission. As demonstrated in Figs.2and3.

Survivalanalysis

Duringdurationofourstudy(30months),numberofevents (death)was36andcensoredcaseswere14(totalnumberof ourcases=50).

Using Kaplan–Meier estimate, the median survival time for all AML patients in the study was 10 months (range:

5.271–14.729months)95%ConﬁdenceInterval.

Mediansurvivaltime inpatients 60 years wasshorter than that in patients <60 years (8ms and 10ms respectively),buttherewasnostatisticallysigniﬁcantdifferenceas

regard mean survival time between both age groups (Log Rank(Mantel–Cox)test:Chi-Square=1.272,p=0.259).

Median survivaltime was statistically signiﬁcanthigher in patients who achieved complete remission (26 months) comparedtothosewhodidnotachievecompleteremission (4months)(LogRank(Mantel–Cox)test:Chi-Square=38.952, p=0.001).ThisdifferenceisillustratedinFig.4.

Median survival time in relapsed patients was shorter than that in non relapsed patients (20ms, 26ms respectively). There was a statistical signiﬁcant difference in mediansurvivaltimeinbothgroups(LogRank(Mantel–Cox) test:Chi-Square=2.609,p=0.050).

MediansurvivaltimeinpatientswithhTERTlevelhigher thancut-offlevelwasshorterthanmediansurvivaltimein Fig.3–ROCcurveshowingcutoffvalueof0.5forRTLasa predictivevalueforAMLoutcome

Fig.2–ROCcurveshowingcutoffvalueof57.57ng/mlfor hTERTasapredictivevalueforAMLoutcome

Fig.4–Comparisonbetweenrespondersandnon- respondersregardingsurvival(p=0.001).CaseProcessing Summary:AMLpatientsinremission:eventsnumber=6, censorednumber=13;AMLpatientsnotinremission:

eventsnumber=30,censorednumber=1

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patients with lower value (5ms, and 14ms respectively).

There was a statistical signiﬁcant difference in median survival time in both groups (Log Rank (Mantel–Cox) test:

Chi-Square=5.653, p=0.017). Similarly, Median survival time in patients with relative telomere length lower than cut-off value was shorter than median survival time in patients with higher value (6ms, and 14ms respectively).

There was a statistical signiﬁcant difference in median survival time in both groups. There was a statistical sig- niﬁcantdifference in mediansurvival time inboth groups (Log Rank (Mantel–Cox) test: Chi-Square=9.305, p=0.002).

ThisisshowninFigs.5and6.

Discussion

The responsetotreatment and overallsurvivalof patients withAMLisheterogeneous.Anumberofprognosticfactors have been described for AML, including age, performance status,andkaryotype.Theseprognosticfactorsareurgently needed in order to be able to better predict treatment outcomesindeﬁnedsubgroupsofpatients[16].

Telomere shortening and telomerase activity has been suggested to be of prognostic value in various human hematopoieticmalignancies[17–19].

In the present study, telomere length and human telomerase reverse transcriptase (hTERT) level were measured in acute myeloid leukemia patients to detect if these parameters might be useful in predicting response to therapyinAMLpatients.

Conventional cytogenetic analysis was done for 18 patientsonly.Chromosomalabnormalitiesweredetectedin 40% of patients (three patients had monosomy 7, one patient had trisomy 21, and one patient had t(15,17)) and 59% of AML patients (thirteenpatients)had normal karyotype pattern. This ﬁnding is in line with the ﬁndings of Mengetal.2013[20].

The patients were treated according to the standard chemotherapy protocol for induction in AML. They were then assessed for response to induction therapy by bone marrow examination at the time of blood count recovery after chemotherapy. They were divided into two groups:

responders, who achieved hematological CR and non- responderswhodidnotachievehematologicalCR.

Allpatientswerefollowedfor30months(thedurationof thestudy).

Although, several studies have reported telomerase expression inacuteandchronicleukemia[17–19].However, uptoourknowledgenolargestudieshaveassayedtelomer- aseinquantitativemanner.

Thepresentstudylookedcomprehensivelyintomeasure- mentoflevelofserumhTERTinAMLpatientsandcorrelated itwithtreatmentoutcome.Sothat,wecouldunderstandthe prognostic role of telomerase and topredict the efﬁcacy of antitelomerasedrugscurrentlyindevelopment.

Inthecurrentstudy,serumhTERTlevelwassigniﬁcantly higher inAML patients compared tocontrolgroup. Thisis consistent with previous studies that reported increased serumhTERTlevelinseveralsolidtumorsasinlungcancer [21, 22],colorectal cancer[23], breastcarcinoma[24], laryn- geal squamouscell carcinoma [25], in gynecological malig- nancies [26], in hepatocellular carcinoma [27], and in hematologic neoplasia [28–30]. As most malignant tumors showed increasedhTERT activity,itmaycontributeaspart of a multistep process, to human carcinogenesis, as it resultsin increasedtelomerase activity.Theresultsof this study ishighly supported by the work of Engelhardt et al.

[19] who revealed signiﬁcantly increased telomerase in diagnostic specimens compared with specimens obtained after treatmentinitiation, which correlatedwiththe disap- pearance of leukemic cells and with the attainment of remission. In addition, Hartmann et al. in 2005 analyzed hTERT different splicing patterns in AML samples, and Fig.6–SurvivalcomparisonaccordingtoRTL(cut-offlevel)

inAMLpatients(p=0.002).CaseProcessingSummary:AML patientswithhigherRTLthancut-off:eventsnumber=10, censorednumber=13;AMLpatientswithlowerRTLthan cut-off:eventsnumber=26,censorednumber=1

Fig.5–SurvivalcomparisonaccordingtohTERTlevel(cut- offlevel)inAMLpatients(p=0.017).CaseProcessing Summary:AMLpatientswithhigherlevelthancut-off:

eventsnumber=19,censorednumber=1;AMLpatients withlowerlevelthancut-off:eventsnumber=17,censored number=13

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telomerase expression was correlated very well with the expressionoftheactivehTERTsplicingvariant.Also,Porika et al. 2011 [31] observed that serum hTERT could have a potential application as a novel biomarker for breast cancerdiagnosis.

As opposed tothe ﬁnding inthe present work,Greiner etal.[32]reportedthat,hTERTmRNAwasexpressedinonly 21%of newdiagnosedAMLpatients[32].Also,Xuetal.[33]

showedthathTERT expressionwasonlydetectableinAML samples with intermediate or high levels of telomerase activity.Thisopposingﬁnding couldbeattributedtodiffer- ent methodologies used. Thus, a direct comparison of the resultsofvariousstudiesisnotpossible.

Inthe present study,the meantelomere length inAML patients was signiﬁcantly shorter, than in healthy control group. This ﬁnding is in accordance withprevious reports [28, 34, 35]. This is supported by the work of Boultwood etal.[36]whoreportedthatprogressivetelomereshortening in patients with chronic myelogeneous leukemia in late chronicphase couldbe linked toup-regulationof telomer- aseactivityanddiseaseevolutioninAML[36].

Regarding age, AML patients <60 years had longer telomerelength than patients60 years butthe difference in telomere length was not statistically signiﬁcant. This ﬁndingisexpectedastelomeres withinhematopoieticcells andothersomatictissuesprogressivelyshortenwithage[8].

However, Hartmann et al. 2005 [28] found signiﬁcantly shorter telomere length in the younger group of patients, their ﬁnding could be attributed to a higher proportion of karyotypicabnormalitiesintheyoungerpatientsthaninthe olderpatientsintheirpopulation.

Astatistically signiﬁcantnegativecorrelationwas found betweenserumhTERT levelandrelativetelomerelengthin the present study. This result isin line with the study of Tukunet al.2006 [37]. Thisobservationcould beattributed tothefactthattelomerasereactivationisarequirement–if notcause–forunlimitedproliferation,whichisanessential characteristicof cancercells. Thisunlimited dividingcapa- city provided by telomerase leads to progressive telomere shortening[37].

SerumhTERTandRTLandresponsetotherapy

In the present study, it was observed that serum hTERT level at diagnosis was significantly lower in patients who achieved complete hematological remission than patients who did not achieve complete hematological remission suggestingthathTERTlevelmayactasapredictivemarker forthetreatmentoutcome.Thisfindingissupportedbythe workof Porikaetal.2011 [31]whosuggestedthatpretreat- ment serum hTERT levels showed a significant correlation withclinicalstageinpatientswithcancerbreast.

Relative telomere length at diagnosis was longer in patientswhoachievedcompleteremissionthaninpatients who did not achieve complete remission. However, the difference did not reach statistical signiﬁcance. Further studieswithlargernumberof patientsare neededtoverify itssigniﬁcance.

Engelhardt [19] observed that, longer telomeres were found after induction chemotherapy in AML and MDS

patients, mostlikelydue tothe lossof the leukemic clone (with shorter telomeres) and the emergence of normal hematopoieticcells(withlongertelomeres)[19].

Serum hTERT level and relative telomere length were comparedbetweenpatientswhosurvivedtilltheendofthe study (totalduration of the studywas 30ms)and patients who died during the study. A highly statistical signiﬁcant difference between both groups as regards mean hTERT level and mean RTL (p=0.001, p=0.001) was found. These observations also suggest that both high hTERT level and short telomere could be considered as poor prognostic factors.

Patientswerefollowedfor30months(thedurationofthe study). No statistically signiﬁcant difference was found between young adult and elderly AML patients in mean survivaltimeusingKaplan–Meierestimate.

Thisresultisnotcomparable tothose obtainedinother studies[38,39]whoreportedhigherincidenceoftreatment- related mortality in elderly group. However, patients who achievedCRhadastatisticallysigniﬁcantyoungeragethan patientswhodidnotachieveCR(t= 2.592,p=0.013).

Theseresultsareinlinewithstudiessupportingoldage ascauseofpoorclinicaloutcomeandpoorprognosticfactor [38,39].

On theother hand,a highstatistically signiﬁcant differ- encewas detectedinmediansurvivaltimeinpatientswho achieved complete remission compared to those who did not achievecomplete remission(26 monthsand 4 months respectively). Thesame was observed in relapsedand non relapsed patients as there was a statistically signiﬁcant difference in median survival time (20 months and 26 monthsrespectively).

Also,survivaltimewasstudiedinrelationtothecut-off value of both hTERT and relative telomere length.

Astatistically signiﬁcantlongersurvivaltimewas observed inpatientswithhTERTlevellowerthanorequaltothecut- offpointandRTLhigherthanorequaltothecut-offpoint.

Targeting the hTERT catalytic subunit as anticancer therapy is theoretically tumor-speciﬁc and might be less toxic due to its speciﬁc expression in tumor and highly proliferating cells compared to other normal cells. Various newly discovered agents represent interesting anti-hTERT candidatesforclinicaldrugdevelopment[40,9].

The telomere and telomerase interactions appear tobe an essential determinant for proliferative capacities of tumor cells. It has been known that telomerase activity provides the ability of proliferation to the malignant cell;

thus, targeting oftumor cellsbyinhibitingtelomerasemay beaneffectivetherapy.

Authors' contributions/Wkład autorów

Accordingtoorder.

Conﬂict of interest/Konﬂikt interesu

Nonedeclared.

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Financial support/Finansowanie

Nonedeclared.

Ethics/Etyka

Thework described inthis article has been carriedout in accordance with The Code of Ethics of the World Medical Association(Declaration of Helsinki)for experimentsinvol- ving humans; EU Directive 2010/63/EU for animal experi- ments;UniformRequirementsformanuscriptssubmittedto Biomedicaljournals.

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