Expression of cancer testis antigens in patients with Hodgkin''''s lymphoma and their clinical correlation Q2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 ART[.]
Trang 1´
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L.M Hodgson Reyesa , ∗, I Olarte Carrillob, C.O Ramos Pe˜ nafielc, A Martínez Tovard,
Q2
E Gallardoe, H Castellanos Sincoe, J Collazo Jalomaf
aHaematology Resident, General Hospital of Mexico, Mexico City, Mexico
bAttending Physician, Molecular Biology Laboratory, General Hospital of Mexico, Mexico City, Mexico
cHead of Clinical Areas, Haematology Department, General Hospital of Mexico, Mexico City, Mexico
dAttending Physician, Molecular Biology Laboratory Head, General Hospital of Mexico, Mexico City, Mexico
eAttending Physician, Haematology Department, General Hospital of Mexico, Mexico City, Mexico
fChief Physician, Haematology Department, General Hospital of Mexico, Mexico City, Mexico
Received8November2016;accepted29November2016
KEYWORDS
Hodgkin’slymphoma;
Testisantigens;
MAGE-Agenefamily;
MAGE-A3expression;
RT-PCR
Abstract
Objective: Todeterminethefrequencyofexpressionofcancertestisantigensandtheirclinical correlationinpatientswithHodgkinlymphoma
Methodology of the study:Inthisanalytical,experimentalandambispectivestudy,theMAGE A-3andNY-ESO-1antigenexpressionwascorrelatedwithclinicalprognosticvariablessuchas clinical stage,responsetotreatment,andrelapse,inatotalof70 patientsdiagnosedwith Hodgkin’slymphomaattheHodgkin’sLymphomaClinicoftheGeneralHospitalofMexico‘‘Dr EduardoLiceaga’’,fromDecember2000toDecember2015.Twenty-fourpatientswere eval-uatedusingRT-PCR,followingextractionofRNA,todetectMAGE-A3andNY-ESO1expression Cellular RNA was extracted from frozen tissue andcontrols usingtrizol (Life Technologies, Paisley, UK).1gofRNA was usedfor cDNA synthesisby M-MLV reversetranscriptase (Life technologies,Paisley,UK)
Results:We studied24 patientswith amedianageof28years,aminimumageof16years andamaximumageof48years,mostlymale.50%ofpatients presentedcompleteresponse
tothefirstlineoftreatmentand27%ofpatientspresentedrelapse,37.5%inrelationtothe expressionofMAGE-A3 ExpressionoftheNY-ESO-1gene wasnotfound inthestudy group Twelvepercentofpatientsdiedduringthestudy,8.33%ofwhomwerealsopositivefor MAGE-A3(p=0.264.95%CI).NosignificantcorrelationwasfoundbetweenMAGE-A3expressionand majorclinicalprognosticvariables
∗Correspondingauthor.
E-mail address:linethhr2@yahoo.es (L.M Hodgson Reyes).
http://dx.doi.org/10.1016/j.hgmx.2016.11.005
0185-1063/© 2016 Sociedad M´ edica del Hospital General de M´ exico Published by Masson Doyma M´ exico S.A This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ).
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Trang 2Conclusion:Although theexpressionofMAGE-A3inthestudy group was37.5% (higherthan reportedininternationalstudies),wefoundnocorrelationwiththemainclinicalprognostics variables.Consideringthattheexpression ofMAGE-A3inthecases studieddoesnotconfer prognosticvalue,makingitimpossibletouseasaprognostictoolinperipheralblood,weare leavingthedoorsopentocontinuewiththislineofresearch,possiblyincreasingthenumberof patientsaswellasprolongingthefollow-uptime
©2016SociedadM´edicadelHospitalGeneraldeM´exico.PublishedbyMassonDoymaM´exico S.A.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons
PALABRAS CLAVE
LinfomaHodgkin;
Antígenos
testiculares;
Familiadegenes
MAGEA;
ExpresióndeMAGE
A-3;
RT-PCR
Expresión de antígenos testiculares de cáncer en pacientes con linfoma Hodgkin y su correlación clínica
Resumen
Objetivo:Determinarlafrecuenciadeexpresióndelosantígenostesticularesdecáncerysu correlaciónconlaclínicaenpacientesconlinfomaHodgkin
Metodología de estudio:Estudiodetipoanalítico,experimental,ambispectivo,Laexpresiónde antígenosMAGEA-3yNY-ESO1fuecorrelacionadaconvariablesdepronósticoclínicotalescomo estadioclínico,respuestaatratamiento,recaída,deuntotalde70pacientesdiagnosticados con linfomaHodgkinen la clínica delinfoma HodgkindelHospital General deMéxico ‘‘Dr EduardoLiceaga’’enelperiodocomprendidoentreDIC2000aDIC2015.Fueronevaluados24 pacientesalosqueselesrealizomedianteRTPCRpreviaextraccióndeRNAlaexpresiónde MAGEA-3yNY-ESO1,elRNAcelularfueextraído deltejidocongeladoydeloscontrolespor mediodetrizol(lifetechnologies,paisley,uk)seutilizo1gdeRNAparalasíntesisdecDNA pormediodelareversotranscriptasaM-MLV(lifetechnologies,paisley,uk)
Resultados: seestudiaron24pacientesconunamedianadeedadde28a˜nos,conunamínima
de16a˜nosyunaedadmáximade48a˜nos,predominiodesexomasculino,50%depacientes presentorespuestacompletaaprimeralíneadetratamientoyun27%depacientespresentaron recaída,enrelaciónconla expresióndelgenMAGEA-3fuede37.5%,noencontrando enel grupodeestudioexpresiónparaelgenNY-ESO1,El12%depacientesfallecieronenelperiodo
deestudio,deestos8.33%fuerontambiénpositivosparaMAGEA-3(p=0.264,95%IC).Nose encontrócorrelacionsignificativaentrela expresióndeMAGEA-3ylasprincipalesvariables clínicaspronosticas
Conclusión:A pesarde quela expresióndeMAGE A-3enelgrupo deestudiofuede 37.5% (mayoraloreportadoenestudiosinternacionales),noencontramoscorrelacionconlas prin-cipalesvariablesclínicaspronosticas,considerandoquelaexpresióndeMAGEA-3enloscasos estudiadosnoconfierevalorpronóstico,porloquenoesposibleutilizarlocomoherramienta pronostica ensangre periférica,dejamos puertasabiertaspara continuar conesta líneade investigaciónconsiderandoelincrementarnúmerodepacientesasícomoprolongareltiempo
deseguimiento
©2016SociedadM´edicadelHospitalGeneraldeM´exico.PublicadoporMassonDoymaM´exico S.A.Esteesunart´ıculoOpenAccessbajolalicenciaCCBY-NC-ND(http://creativecommons
Hodgkin’s lymphoma (LH) is a neoplasm characterised
by lymphatic infiltration of both Hodgkin cells and
Reed-Sternbergcellssurroundedbyaninflammatorycellinfiltrate
composedofplasmacells,eosinophilsandhistiocytes.5
Fac-torsassociatedwithitsonsethavebeendescribedasfamilial
factors, immunosuppression and association with viruses,
the association with Epstein Barr virus being reported in
morethan50%ofcases.6
Themajorityofpatientspresentwithlymphadenopathy,
with cervical lymphadenopathy being the most frequent,
followed by axillary and, less frequently, inguinal
lym-phadenopathy,extra-nodalmanifestations,eitherbydirect
invasionorhaematogenousdissemination,morefrequently involving the spleen, lung and liver, and less frequently involvingthebonemarrow.Systemicsymptomsoccurinup
toone third of patients, and include fever, nightsweats, weightlossandchronicitching
Thediagnosisismadebyexcisionalbiopsyoftheaffected lymphatic tissue.Riskstratificationis performedusingthe AnnArborstagingsystembasedontheinvolvedlymphnode area, the presence or absenceof bulky mass, extranodal diseaseandpresentBsymptoms
ThestandardoftreatmentincludestheuseoftheABVD chemotherapyregimen(doxorubicin,bleomycin,vinblastine
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Trang 3and dacarbazine) with or without 20 -30Gy radiotherapy
(RT), achieving 98% survival rates Other regimens are
associatedwithincreasedtoxicity(BEACOPP).8Factorsfor
determiningtherapy includethehistologicaltype,clinical
stage(poorprognosisIIIandIV,presenceofbulkymassand
constitutionalsymptoms)
The role of PET-CT (positron emission tomography) in
patientswithHodgkin’slymphomaissignificant initiallyin
staging(intervalPET-CT)forthemostaggressive
therapeu-ticdecision,aswellasthe PET-CTatendof treatmentto
evaluatetheresponsetotreatment(totalresponse,partial
response, stabilityof the disease, progressionor relapse)
andtodeterminewhetherornottoaddconsolidation
radi-ationtherapy.7Itscostisalimitationfortheuseofitinour
environment
Overall post-treatment survival is good Late
toxic-ity associated with treatment is the main disadvantage,
withsecondarymalignancies(myeloidleukaemia,
myelodys-plastic syndrome, lung cancer and breast cancer) and
cardiovascular diseases (leftventricular failure and
coro-naryarterydisease)beingthemostfrequent.Post-relapse
alternatives include high-dose chemotherapy, autologous
haematopoietic stem cell transplantation, andthe use of
monoclonalanti-CD30antibodies
Since 1998 the International Prognostic Index (IPI),
or Hasenclever index, has been useful in identifying
populations at risk of therapeutic failure, including
clin-ical and biochemical parameters (albumin<4g/dl, stage
IV, age >45 years, haemoglobin<10.5g/dl, white blood
cells>15×103/l and lymphopenia, masculine gender),
classifyingprogression-freesurvivalat5yearsaccordingto
thenumberofriskfactors:0factors:84%,1factor:77%,2
factors:67%,3factors:60%,4factors:51%,5factors:42%
The lactate dehydrogenase test linearly reflects the
tumour load Despite advances in imaging studies with
regard to Hodgkin’s lymphoma, there are few molecular
markers usedto identifypopulations at risk of treatment
failureor forfollow-up ofresponsetotreatment Current
knowledgeabout theexistenceofcirculating tumourcells
attributesupto90%ofcancer-relateddeathstometastatic
diseaseinsolidtumours
Tumour cells must pass through the endothelium and
circulate through the bloodstream until they are either
eliminated by immune response mechanisms or find an
appropriate microenvironment in which they reside in a
latent state, and eventually acquire the ability to
prolif-erate at a later time.Only one out of 10,000 circulating
cancer cells is able toform metastases(HALLMARK 2013,
Fig.1)
The clinical utility of circulating tumourcells depends
ontheir availabilityinperipheral blood, whichallows the
studyoffundamentalaspectsinthediagnosis,stagingand
prognosisofmalignancies,methodsbasedonthedetection
of free circulating DNA, RT-PCR and quantitative RT-PCR,
demonstratinggreatersensitivitywhencomparedwithflow
cytometry
There are approximately 100 cancer testis antigens
(CTAs) These antigens aremolecules which are normally
expressedingermcells.MAGE-A3isaCTAwhoseexpression
is restricted to germ cells in the testis (primary
sper-matocytes, spermatogonia and trophoblasts), abnormally
expressingindiversetumourcells.10
Sustaining proliferative signaling
Evading growth suppressors
Avoiding immune destruction
Enabling replicative immortality
Tumor-promoting inflammation Activating
invasion &
metastasis
Inducing angiogenesis
Genome instability &
mutation
Resisting cell death
Deregulating cellular energetics
Figure 1 Hallmark 2013 This metastasis capacity of solid tumourshasbeen describedinlymphomas These circulating tumourcellsmustpassthroughseveralphases,including circu-lationtotargetorgans,proliferation,evasionoftissueimmune responseandovercomingmetabolicdifficulties
MAGE-A was initially identified in melanoma All the genesof the MAGE-Afamily have aprotein encodedby a singleexon, preceded by several non-coding exons They belongtoagroupcalledMAGEclassI,havingsimilar func-tionsandcharacteristics (60 -98%similarityintheircoding sequence)(Fig.2)
TheMAGEtypeIfamilyincludesthesubgroupsMAGE-A, MAGE-BandMAGE-C.Theyareexpressedintumourcellsof thelung,head,neck,bladderandbreast,aswellasgastric cancer, colorectal cancer, and haematologic malignancies suchaslymphoma,leukaemiaandmyeloma,amongothers The MAGE type II family includes the subgroups MAGE-D, MAGE-E,MAGE-F, MAGE-G, MAGE-H, MAGE-I2 and Necdin, whicharecoded bygenes present inthe X chromosomes, describedintheq28(MAGE-Agenes),p21.3(MAGE-B),q26 (MAGE-C),andP11(MAGE-D)regions.14Thefunctionofthese antigensisnotfullyknown.Ithasbeenwelllinkedto pro-teinubiquitinationprocessesincludingAMPK(AMP-activated proteinkinase),whichhascriticaltumoursuppressing activ-itiesinbothhumansandexperimentalmodels,sothatbeing ubiquitinatedcanpromotedelayedtumourgrowththrough stimulationofthemTORpathway,inhibition ofautophagy, and stimulation of metabolic activity Its anti-apoptotic properties(inhibitsp53)mayexplainthepersistenceof min-imalresidualdiseaseinsomemalignantcancers,including Hodgkin’slymphoma,wherethe potentialfor cureisvery high
The frequency of expression is highly variable in dif-ferentcancertypes, and cancersconsidered tohave high expression of these antigens include: melanoma, ovarian cancerandlungcancer,whilesomehaematopoietic malig-nancies,kidney,colonandpancreaticcancerareconsidered
tohave low frequency of expression.15,16 Exceptionshave beenreportedregardinghaematologicmalignancies,suchas
inmultiplemyelomawheretheexpressionofthe CT7/MAGE-C1antigenishigh,asistheexpressionofCT45inHodgkin’s lymphoma
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Trang 4Chromosome X
Genome
5º mRNA
1aa Protein
DNA (-)
Exon 3
945 bases
116aa
GTA
Negative strand transcription
98aa
AGT 3º
314aa MAGEA3 COOH-terminal region MAGE homology domain (MHD) MAGEA3 NH2-terminal region
286aa
Figure 2 MAGE-A3is thethirdmember ofthe tumour antigenfamily Itsexpressionisrestricted tonormaltesticular tissue andplacentaltrophoblast cells,andaberrantexpressioninseveraltypesofcancer.BiswajitDas, 2014-04atlasofgeneticsand cytogeneticsinoncologyandhaematology
As for immune recognition, the A3 melanoma antigen
family (MAGE-A3) was the first human tumour-associated
antigenfoundtobespecificallyrecognisedbyCD8+T-cells
MAGE-A3iscoupledtoMHCclassI.Theabsenceof
expres-sionin normaltissuesassuresanimmune responseagainst
the tumour Other cancer testis antigens are the BAGE,
GAGE,LAGEandNY-ESO-1genes,whichtogetherwithMAGE
arealsorecognisedinvariouscellstransformed
neoplasti-Q3
callybyCD8(+)T-lymphocytes.29 ,33,39
MAGE gene expression is regulated through a
methyl-ation mechanism.12 Its expression is inhibited in normal
tissueby epigeneticmechanismsoftranscriptional
repres-sion through hypermethylation of the promoter gene Of
the known cancer testis antigens, those most frequently
expressed in tumours are MAGE-A1, MAGE-A3, NY-ESO-1,
SSX-2ySSX-4
NY-ESO-1inducesspontaneousimmuneresponsesofthe
host in up to50% of the patients withmalignancies that
expressit.Itisconsideredoneofthemostimmunogenic.34,35
Being an important candidate for immunotherapy,
ini-tially identified in oesophageal cancer due to its name,
its expression has been demonstrated by mRNA reverse
transcriptase polymerase chain reaction (RT-PCR) in 30%
of melanoma, lung, and bladder cancers, 42% of breast
cancersand 65% of medullary thyroid carcinomas, among
others A direct relationship between NY-ESO-1, tumour
evolution and antibody titre has been observed,
demon-stratingitsabilitytoinduceimmuneresponsemediatedby
both CD4+ and CD8+ lymphocytes.31,32 Its use is known in
variousimmunotherapiesagainstcancer,suchasmalignant
melanoma,inwhichtherecombinantNY-ESO-1proteinwas
injectedintradermally.34,35,36
There have been studies that evaluated the
expres-sionoftheseantigens(MAGE-A3)indifferenthaematologic
malignancies.In 2010, Han et al reported the detection
of circulatingtumour cells in patients withnon-Hodgkin’s
lymphoma, using MAGE-A3 gene expression in peripheral
blood, withexpression in 47.3% of patientsstudied (total
of 95 patients), finding no relation to survival A
reduc-tionofMAGE-A3expressionwasreportedfollowingeffective
chemotherapy, suggesting the MAGE-A3 geneas a tumour
marker in non-Hodgkin’s Lymphoma, without conferring prognosticvalue.1
In2011Inaokaetal.reported,inaBrazilianpopulation (totalof38patients),theexpressionoftheMAGEfamilyin 21%ofpatientswithHodgkin’slymphoma.Consideringthis expression asatarget forimmunotherapy, expressionwas evaluatedfor theMAGE-A family(18%), aswell as MAGE-C1/CT7 (13.2%), MAGE-C2/CT10, NY-ESO-1 and the GAGE family, withthefirst twobeingthemost frequently asso-ciatedwithanadverseprognosis.12
MAGE-A3 is considered a promising tumour-associated antigen for the selective targeting of malignant cells with immunotherapy Its usefulness has been mentioned
in patients with post-transplant multiple myeloma, using MAGE-A3/Poly-ICLC immunisation followed by adoptive transfer of autologous T-cells from prepared and co-stimulated vaccines In a phase II clinical trial, T-cell infusionswerewelltolerated.90%ofpatientshadlocal reac-tionsatthevaccinesite.Overallsurvivalat2yearswas74% (95% CI,54 -100%) and56% had2 yearsof event-free sur-vival(95%CI,37 -85%).AhighfrequencyofT-cellresponses
tothespecificvaccinewasconcludedinthiscase.28Similar resultswerereportedinaphaseIIclinicaltrialusing antigen-specific immunotherapy to generate an immune response againstMAGE-A3,toeradicate itsexpression bytumourin
182 patients withsmall celllung carcinoma The average follow-upwas44months.35%ofpatientsreceivinga recom-binant MAGE-basedvaccine showedclinicalbenefitsbased
onimmunotherapy
In theHaematology Department at the General Hospi-tal of Mexico, a line of research intogenes of the MAGE familywaspursuedforpurposesofmonitoringand prognos-ticevaluationinpatientswithhaematologicmalignancies
In2005 astudywasconductedandpublishedby DrRozen FullerE.onMAGE-Ageneexpressionanditsprognostic cor-relationinacutelymphoblasticleukaemia,withatotalof47 patients,reportingtheexistenceofaprognosticrelationship betweenthediseaseandthepresenceofMAGE-A3.In2007,
anewstudywasconductedbyDrOlarteetal.,evaluating MAGE-A3expressioninpatientswithdiffuselargeB-cell lym-phoma.Withatotalof28patients,astatisticallysignificant
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Trang 5advancedstages, high LDHlevels,poorresponseto
treat-ment and lower survival.2 In 2015, Mendoza Salas et al
studied thefrequency ofcancer testisantigens inchronic
myeloidleukaemia(CML).Atotalof10samplesfromhealthy
individualsand65bonemarrowsamplesfrompatientswith
CML were analysed, reporting a presence of MAGE-A3 of
32.3%, with MAGE-A4 having a greater presence of up to
63%.3In2016DelaCruzRosasetal.reportedthepresenceof
MAGE-A3in21%ofpatientswithmultiplemyeloma,
associ-atingitwithresistancetotherapy,progressionandreduction
insurvival
Wehaveoutlinedtheimportanceofknowingthe
expres-sion of cancer testis antigens in different haematological
malignancies, as tumour markers, prognostic factors and
targetsforimmunotherapyindifferenthaematological
neo-plasms.Wehaveproposedthisstudybecausetheexpression
of the cancer testis antigens MAGE-A3 and NY-ESO-1 in
patients with Hodgkin’s lymphoma in Mexico is unknown
General objective:Todetermine thefrequency of
expres-sion of the cancer testis antigens MAGE-A3 and NY-ESO-1
and their clinical correlation in patients diagnosed with
Hodgkin’s lymphoma in the Hodgkin’s lymphoma clinic of
theGeneralHospitalofMexico‘‘Dr.EduardoLiceaga’’from
December2000toDecember2015.Methodology:Ourstudy
isanalytical,experimentalandambispective.Independent
variables: Expression of MAGE-A3 andNY-ESO-1 Variables
dependentonclinicalprognosis:Clinicalstage,responseto
treatment, relapse.A totalof 24 patients wereincluded,
fromwhomperipheralbloodsamplesweredrawnwithprior
informedconsent
Weincludedpatientsovertheageof16,with
histopatho-logical (lymph node) diagnosis of Hodgkin’s lymphoma
corroboratedbyimmunohistochemistry(Cd15,Cd30,Cd20,
andEBVmarkers)intheindicatedperiod.Informedconsent
wasobtained for thedrawing of peripheral blood for the
study.We excludedpatients whowerenot followedupin
ourcentreandeliminatedpatientswhoseperipheralblood
sampleswereinsufficientfortheexpressionofthegenes
Hypothesis:Iftheexpressionofthecancertestisantigens
MAGE-A3andNY-ESO-1isfound inpatientswithHodgkin’s
lymphoma,thenthismayberelatedtorelapseand
treat-mentfailurewithinashortfollow-uptime
Expression of the genes was evaluated by reverse
transcriptasepolymerasechainreaction(RTPCR),with
tes-ticular tissue being our positive control, to assess mRNA
expressionofcancertestisantigens:K562CellLinederived
fromapatientwithchronicmyeloidleukaemia(CML).Our
negative controlconsistedofperipheralbloodsamplesfrom
healthydonors,fromwhichweobtainedmononuclearcells
(lymphocytes,monocytes,blasts)usingtheFicoll-Hypaque
gradientmethod
The mononuclear cell separation procedure was done
with the Ficoll-Hypaque gradient method with a Ficoll
density of 1.077g/cm3; mRNA isolation was performed
by alkaline lysis using the Trizol/Invitrogen reagent;
integrity (2% agarose gel) was carried out by
molecu-lar biology expertsfrom the molecularbiology laboratory
area of the General Hospital of Mexico The
quantifi-cation and purity of the RNA and detection of cancer
testisantigensby polymerasechainreaction,aredetailed
below
Quantification and purity. Nucleic acids efficiently absorb ultraviolet light due to the presence of aromatic nitrogenous basesalong the DNA strands The UV absorp-tionofDNAisacharacteristicofthemolecule,whichisused efficientlytodetermineitsconcentration.Eachofthebases hasitsownuniqueabsorptionspectrumandtherefore con-tributesdifferently tothetotalpropertyof UVabsorption
ofaDNAmolecule
Theabsorbenciesusedare260nmand280nm.At260nm the nucleic acids reach their maximum light absorption Proteinshaveamaximum absorptionat280nm(mainlyby tryptophanresidues).Readingsatthiswavelengthcanshow
if there is protein contamination The calculation of the A260/A280ratioisacommonwaytoexpressthepurityof geneticmaterial.Dependingonthenucleiccomposition,a valueof1.65 -1.9indicatesapuresample
The cDNA of healthy donors, as well as that of patients withlymphomaandcontrols,wassynthesisedfrom2gtotal RNA.ThefinalcDNAvolumewas20g.TheRNAwasmixed with1lofoligo(dT)12 -18primer(INVITROGEN,Carlsbad, CA)and1lof10mMdNTPs(Applied Biosystems,Roche) Themixturewasincubatedat65◦Cfor5minutesandplaced
onice Weadded4lof5×buffer (Tris -HCl250mM,KCl
375mMMgCl2 15mM),2lof DTT(0.1M), andthe corre-spondingvolumeofH2O,andthemixturewasincubatedat
37◦Cfor2min.Then1lofM-MLVRT(200U)(INVITROGEN, Carlsbad,CA)wasaddedandthemixturewasincubatedat
37◦Cfor50min.Theenzymewasinactivatedbyincubating
at70◦Cfor15min
ForthePCR,afinalvolumeof10lwasused,inwhich
10mMTris -HCl,50mMKCl,1.5mMMgCl2, 100MdNTPs, 0.2U Taq DNA polymerase, (INVITROGEN, Carlsbad, CA) 0.5Msenseprimer,0.5Mantisenseprimerand1lcDNA weremixed.TheprimersfortheMAGE-A3andNY-ESOgenes were described in Olarte, 2012 The same sequence of primerswasusedtoamplifythesamplesofpatientsatthe GeneralHospitalofMexico.Asacontrol,theGAPDH consti-tutivegenewasused.Thesequenceofprimersusedaswell
astheexpectedlengthofthePCRproductcanbefoundin Table1
Standardisation was carried out using various concen-trations of primer and alignment temperatures, and was performed with testicular tissue, and the K562 cell line, whicharethepositivecontrols.Thesameamplificationwas corroboratedinhealthydonorswheretherewasno amplifi-cationofgenes
Thisstudycomplieswithinstitutionalethicalstandardsand theGeneralHealthLawonhumanexperimentation,aswell
astheDeclarationofHelsinki,withamendmentbythe Gen-eralAssemblyofTokyo,Japanin1983
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Trang 6Table 1 Sequenceofprimers.
Length725basepairs
Conditions:96◦C -1min
60◦C -45s
30cycles
Length307basepairs
Conditions:96◦C -1min NY-ESO-1REVERSE CTGGCCACTCGTGCTGGGA
67◦C - 45s
72◦C - 1min
30cycles
Statistical analysis
StatisticalanalysiswasperformedusingtheSPSSsoftware,
version 2.0 Initially, descriptive statistics were used to
establish means, medians and ranges The Pearson
cor-relation coefficient was established between qualitative
variables (lactate dehydrogenase and RQ-PCR for
MAGE-A3and NY-ESO-1, etc.) The chi-square test wasused for
the hypothesis test between the MAGE-A3 and
NY-ESO-1 expression and was considered significant at or below
0.05(95%CI)
Results
A totalof 24 patients were analysed, with malepatients
predominatingat66%,withanaverageageof28years.The
predominanthistologicalstrainwasthemixedcelltypein
66%,followedbynodularsclerosisin29.1%,and4%richin
lymphocytes
The diagnosis ofclassical Hodgkin’slymphomarequires
theidentificationofReed-Sternbergcellsintheappropriate
cellularenvironmentandwiththeirown
immunohistochem-ical characteristics Reed-Sternberg cells are positive for
CD30andCD15,negativeforCD45andEMA,andsometimes
positivefor Bmarkers Whatis expectedin terms of
pos-itiveexpression is a predominance of CD30 against CD15
ThepresenceofCD15,CD30andCD20wasevaluatedinour
immunohistochemicalstudy.CD30wasthemostfrequentin
71%ofpatients,followedbyCD15,withCD20positiveinone
thirdofpatients.ThepresenceoftheEpsteinBarVirus(EBV)
correspondedto50%ofpatients
With regard to clinical stages, 66% were in advanced
stages(IIIandIV).WiththeHasencleverprognosticindex,
weevaluatedvariableswithanadverseprognosticimpact
Wedividedthescoresintotwogroups:the0 -2grouphad
a 5-year progression-free survival rate greater than 67%,
while the 3 -5 group had a survival rate of less than 60%
at5 years.We found that33% willhave an adverse
prog-nosiswithacalculatedsurvivalrateoflessthan60%.LDH
numbersaredirectlyrelatedtotumoursize.Figuresgreater
than250mg/dlrepresentanadversemanifestationinterms
ofprognosisandfollow-up,beingariskfactorforrelapse
Themajorityofourpatients,54%,presentedfiguresgreater
than250mg/dl
TheABVDchemotherapy regimenwasusedasfirstline treatmentin87%ofpatients.Onlyonepatientreceivedan initial regimen with MOPPand 2 patients did not initiate anytreatmentregimenduetotheirclinicalconditions.Fifty percentreceivedradiationtherapy.Fiftypercentshoweda completeresponsetothefirstlineoftreatment,evaluated withPETCTatthe endof treatment,recording arelapse percentageof27%
The expression of MAGE-A3 was37.5% in our patients
InrelationtoNY-ESO-1intheanalysisoftestisantigensby PCR,noexpressionwasfoundinanypatientfor thisgene WhentheMAGE-A3expressionwascorrelated,themain clin-icalvariableswere:response totreatment(p=0.224, 95% CI),Hasencleverprognosisscore(>2 or<2)(p=0.371, 95% CI),clinicalstage(p=0.689,95%CI)andrelapse(p=0.807, 95%CI).Nostatisticallysignificantcorrelationswerefound Sixty-sixpercentoftheMAGE-A3positivecaseswere posi-tiveforEpsteinBarand44%alsopresentedhighLDHfigures,
asshowninTable2
Of all patients, 12% (3 patients) died in the follow-up period.ThemajorityofthesewerealsopositiveforMAGE-A3
(p=0.264,95%CI),althoughitwasnotstatistically signifi-cant,mostlikelyduetothesmallnumberofpatients
TheAmericanCancerSocietyhasestimated8500newcases
of Hodgkin’s lymphoma As of 2004, there were 935 new casesinMexicowithahigherincidenceinmales.Interms
ofdescriptivestatistics,wecouldobservesome characteris-ticsofourstudygroupassociatedwithanadverseprognosis, amongwhichwementionedLDHlevelsabove250mg/dl(the higher value,the largerthe tumoursize), absenceof the CD15markerandadvancedclinicalstageatdiagnosis (clin-icalstagesIIIandIV),withacalculatedsurvivalrateofless than60%at5yearsaccordingtotheinternationalprognostic index(Hasencleverindex).20
MostofthepatientsreceivedtheABVDregimen(the cur-rent standard treatment regimen) asfirst-line treatment, andhalfofthemalsoreceivedradiationtherapy.Thefinal responsetotreatmentwascomplete in50%ofcases.This figureisfarfromwhatweexpected,ifweconsidertheideal uptake target (80%) ofthese patients in alocalised stage (stagesI -II)forasurvivalrategreaterthan92%.21,25
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Trang 7Table 2 CorrelationofprognosticvariableswithMAGE-A3inpatientswithHodgkin’slymphoma.
Negative Positive Pearson’schi-squaredtest
Degreeoffreedom Count %ofNintable Count %ofNintable Significance
Strain
RT
1 0.91575915
Response
4 0.22495865
HPS
1 0.37109337
IntheUnitedStatesandEurope,theratioofHodgkin’s
lymphoma to the Epstein Barr virus is 40% What is
reported in Mexico does not differ greatly from
interna-tionalreports.21Thepredominantstrainisnodularsclerosis
in more than 60% of cases Previous reports in reference
centresinMexicomatchourdata.Itshouldbenotedthatin
2011,mixedcellularitywasreportedbytheNationalCancer
Institute (INCAN)and the Salvador Zubirán National
Insti-tuteofMedicalScienceandNutrition(ICMNNSZ)asthemost
frequentlyfoundstrain.19
MAGE-A3expressionwasfoundin37.5%ofourpatients,
whichwashigherthanthatreportedininternational
stud-ies, such asthe 2011 study by Dr Riguel J Inaoka at the
Departmentof Pathology of the Federal University of Sao
Paulo;in38patientswithHodgkin’slymphomaalowerrate
ofexpressionthanourswasreported,at20%.Itshouldbe
noted that in their cases they found greater positivityin
advancedstagescomparedtotheinitialstages ofthe
dis-ease,coincidingwithourstudy.23
InrelationtoNY-ESO-1intheanalysisoftestisantigens
byPCR,thefactthatitsexpressionwasnotfoundmaybe
relatedtothefactthatitismoreofanintracellularmarker
Therefore,itsidentificationismuchmoreprobableinsolid
tumourssuchasoesophageal,liposarcomaandothers.23
The estimated mortalityofHodgkin’sLymphomain the
United States is 1300 cases each year The majority of
patientsdiagnosedwithHodgkin’slymphomaareexpected
tohave disease-free survival exceeding80% at 5 yearsof
follow-up.Theone-yearsurvivalrateforallpatients
diag-nosedwithHodgkin’sdiseasereaches92%,with5-yearand
10-year survival rates being around86% and 80%,
respec-tively,and themortalityrate inour studydoes not differ
fromwhatisexpectedattheinternationallevelandisclose
tothenationallevel.InastudyconductedattheINCMNSZ,in
156patientsstudiedatotalof15fataleventswerereported, withcompleteremissionin61.6%ofpatients.25
No significant correlation was found between MAGE-A3expression andthemajor clinicalprognosticvariables Therewas atrend associating theexpression ofMAGE-A3 cancertestis antigens withmortality,advanced stageand relapse,buttheassociationwasnotstatisticallysignificant Therefore,wecouldnotuse itasaprognostictoolinour patients with Hodgkin’s lymphoma The ideal prognostic markerincancerisonethatallowsustoaskquestionssuch as:Canthepatientachievecancerremissionornot?What
isthe besttreatment? What wasthetreatment response? Couldthe patient have a relapseafter having achieveda completeresponseandcanthisbeanticipatedbefore clini-calsignsbecomeevident?Therefore,anidealmarkermust
behighlyspecificforaparticulartumour,itmustallowthe detectionofcancerevenwithhiddendisease,i.e.itshould
bepresent evenin theearlystages,andlastly,itmustbe verysensitivetoavoidfalsenegatives.Inourstudy,MAGE-A3 andMY-ESO-1didnotfullycomplywiththese characteris-tics
Conclusions
The expression of cancer testis antigens (MAGE-A3) was 37.5%in peripheral blood in patients withHodgkin’s lym-phoma No statistically significant relationship was found between MAGE-A3 and clinical prognostic parameters in patientswithHodgkin’slymphoma.Itisnotpossibletouse
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Trang 8Weconsideritimportanttostudytheexpressionof
MAGE-A3antigensinourpopulationwithmalignantneoplasms,in
particularHodgkin’slymphoma.Werecommendcontinuing
withthislineofresearchandopeningthedoorstonew
stud-iesthat increase the numberof patientsand prolong the
follow-upperiods
Protection of human and animal subjects.The authors
declarethat the proceduresfollowed were in accordance
withtheregulationsoftherelevantclinicalresearchethics
committeeandwiththoseoftheCodeofEthicsoftheWorld
MedicalAssociation(DeclarationofHelsinki)
Confidentiality of data.Theauthorsdeclarethattheyhave
followedtheprotocolsoftheirworkcenteronthe
publica-tionofpatientdata
Right to privacy and informed consent.Theauthorshave
obtainedthe written informed consentof the patients or
subjectsmentionedinthearticle.Thecorrespondingauthor
isinpossessionofthisdocument
Funding
This study was conducted with the support of the
Army of Nicaragua, the Government of Mexico through
the fellowship of excellence granted through the
Sec-retary of Foreign Affairs, and the support of CONACYT
with project number 162269, as well as the Research
Directorate of the General Hospital of Mexico with
reg-istrationnumbers DIC/09/04/03/131,DIC/08/204/04/017,
DIC/12/204/05/01
Q4
Theauthorsdeclarethattheyhavenoconflictofinterests
Q5
4,9,11,13,17,18,22,24,26,27,30
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