w w w . j c o l . o r g . b r
Journal
of
Coloproctology
Original
Article
Analysis
of
neutrophil–lymphocyte
ratio
as
a
prognostic
element
in
the
response
to
neoadjuvant
therapy
in
rectal
cancer
Leonardo
Vieira
Polli
a,b,c,∗,
Mauro
Pinho
a,daPostgraduateHealthandEnvironmentProgram,UniversidadedaRegiãodeJoinville(UNIVILLE),Joinville,SC,Brazil
bServicedeOncology/Radiotherapy,HospitalMunicipalSãoJosé,Joinville,SC,Brazil
cHospitalSãoJosé,JaraguádoSul,SC,Brazil
dDepartmentofSurgery,HospitalMunicipalSãoJosé,Joinville,SC,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received1September2014 Accepted28September2014 Availableonline28January2015
Keywords: Neoplasia Rectum Radiotherapy Lymphocytes Chemotherapy
a
b
s
t
r
a
c
t
Introduction:Thepreviousradio-chemotherapyapproachishighlyrelevantinthe manage-mentofrectalcancer,collaboratingonorganfunctionalpreservation,beingperformedprior tosurgery.Theinflammatoryresponseplaysanimportantroleinthistreatment.
Objective:It consistsincorrelatingthenumber ofperipherallymphocytesandthe
neu-trophil/lymphocyte ratioin the peripheral blood with tumor response to neoadjuvant
therapy.
Methods:ReviewofmedicalrecordsofpatientswithrectalcancerinHMSJandHSJOncology Servicessince2009–casessubmittedtoneoadjuvanttreatmentwithradio-chemotherapy. Results:Ofthose96patientswiththisdiseasewhounderwentneoadjuvanttherapywith
radio-chemotherapy, 35 patients wereeligible;complete tumor responsewasobserved
in 11 cases(31%), and 9 weresubmitted to surgical treatment. Comparing the
leuko-cyteparametersbetweenpatientswithcompleteresponse(CR)andincompleteresponse (IR)thefollowingvalueswereobserved:totalnumberofleukocytes(mean)CR7390.9×IR 7220.4(p=0.8);totallymphocytesCR2103×IR1960.9(p=0.4);neutrophil/lymphocyteratio CR3.55×IR3.79(p=0.5).Themeanradiotherapydosewas49.1Gy,withCR47.3×IR50.0 (p=0.06).
Conclusion: It was not possible to demonstrate in this study a significant relationship betweencompletetumorresponsetoneoadjuvanttherapywithrespecttobloodleukocyte parametersanalyzed.
©2015SociedadeBrasileiradeColoproctologia.PublishedbyElsevierEditoraLtda.All rightsreserved.
∗ Correspondingauthor.
E-mail:Leonardo.polli@univille.br(L.V.Polli),drpolli@hotmail.com(M.Pinho).
http://dx.doi.org/10.1016/j.jcol.2015.01.003
Análise
da
relac¸ão
neutrófilo-linfócito
como
elemento
prognóstico
na
resposta
à
terapia
neo-adjuvante
no
câncer
do
reto
Palavras-chave: Neoplasia Reto Radioterapia Linfócitos Quimioterapia
r
e
s
u
m
o
Introduc¸ão:Aabordagemradio-quimioterápicapreviaapresentagranderelevânciano manu-seiodocâncerdereto,colaborandonapreservac¸ãofuncionaldoórgão,sendorealizada previamenteàcirurgia.Arespostainflamatóriatempapelimportantenestetratamento. Objetivo: Consisteemcorrelacionaronúmerodelinfócitosperiféricosearelac¸ão neutrófi-los/linfócitosnosangueperiféricocomarespostatumoralàterapianeo-adjuvante. Métodos: Revisãodeprontuáriosdospacientesportadoresdecâncerretaldosservic¸osde OncologiadoHMSJeHSJ,desde2009,casossubmetidosaotratamentoneo-adjuvantecom radio-quimioterapia.
Resultados: Dototalde96pacientesportadoresdestaenfermidade,submetidosàterapia neo-adjuvantecomradio-quimioterapiaforamelegíveis35pacientes,tendosidoobservada
respostacompletatumoralem11casos(31%),enoveforamsubmetidosaotratamento
cirúrgico.Nacomparac¸ãodosparâmetrosleucocitáriosentreospacientescomresposta completa(RC)erespostaincompleta(RI)foramobservadososseguintesvalores:número totaldeleucócitos(média)RC7390,9×RI7220,4(p=0,8);linfócitostotaisRC2103×RI1960,9 (p=0,4);relac¸ãoneutrófilo/linfócitoRC3,55×RI3,79(p=0,5).Adoseradioterápicamédiafoi de49.1Gy,sendoRC47,3×RI50,0(p=0,06).
Conclusão: Não foi possível demonstrar no presente estudo relac¸ãosignificativa entre respostacompletatumoralàterapianeo-adjuvantenosparâmetrosanalisadosdoperfil leucocitário.
©2015SociedadeBrasileiradeColoproctologia.PublicadoporElsevierEditoraLtda. Todososdireitosreservados.
Introduction
Thegoodresultsobtainedwiththeassociationof
chemother-apy with radiotherapy led to the adoption of neoadjuvant
therapy inthe treatment ofrectal cancer,with the aimof
promotingareductionintumorsize,allowingbetter
condi-tionsofresectabilityandsphincterpreservation;thishasled
someauthorstosuggestanon-surgicaltreatmentincasesof
completeresponsetoneoadjuvanttherapy.Itwasnotthusfar possibletoidentifyprognosticfactorsthatcouldcontributeto theprognosis,withhighlevelsofaccuracy,totheoccurrence
oftumorregressioninresponsetoneoadjuvanttherapy.
Onthe other hand,several studies havesuggestedthat
thehostimmuneresponseplaysaveryimportantroleinthis
response.Variousaspectsofimmunologyhavebeenthe
sub-jectofstudies,takingintoaccountfactorsrelatedtothetumor, thehostandtheirinteraction.Amongthesefactors,themost consistentresultshavebeenobtainedintheevaluationoflocal
andsystemiclymphocyticresponse,characterizedby
lympho-cyteinfiltrationintothetumortissueandbyagreaterpresence
oflymphocytesinperipheralblood.Theprognosticvalueof
thisgreatersystemiclymphocyticexpressionhasbeenshown
bystudiessuggestingabettertumorresponsetoneoadjuvant
therapyinpatientswhohaveanincreasednumberof
lympho-cytes,inproportiontothetotalnumberofneutrophils,and
thisrelationshipissummarizedbytherelationshipbetween
neutrophilsandlymphocytes.1
Therefore,the aimofthisstudy istoanalyzeapossible
prognosticvalueofbloodleukocytesinperipheralbloodwith
tumorresponsetoneoadjuvanttherapy,observedinagroup
ofpatientswithrectalcancer.
Methods
Patients
Medicalrecordsof96patientswithrectalcancer,treatedin
theRadiotherapyServicesoftheHospitalMunicipalSãoJosé
(HMSJ)inJoinvilleandoftheHospitalSãoJosé(HSJ)inJaragua
doSul,werereviewed;thesepatientsunderwentneoadjuvant
treatmentwithradiotherapyandchemotherapy.
Amongtheseparticipants,thosewithincompletedatain
their medicalrecords (makingit impossible toassess their
outcomes)wereexcluded, aswellaspatientswithno
com-pletebloodcount(CBC)availableforevaluation,patientswho
did notconcludethe radiochemotherapy,or who were lost
tooncologicalfollow-up.Thus,35patients(14males)witha meanageof55yearswereeligible.
Neoadjuvanttherapywasindicatedforpatientswith
rec-tal adenocarcinomaslocatedinthe middleand lowerthird
of the rectum, whose staging suggested parietal invasion
(T3 or T4 stage),or bythe presenceof a possible
perirec-tal lymphadenopathy. All patients underwent proctologic
examination, endoscopic assessment and biopsy through
colonoscopyorrectosigmoidoscopy,andanimaging
exami-nationbycomputedtomographyormagneticresonancewas
Neoadjuvanttherapytechnique
All patientsunderwent two-dimensional radiotherapy with
simulationsusingcontrast-enhancedradiographyofthe
rec-tum and bladder, or three-dimensional radiotherapy with
simulations using computed tomography and intravenous
contrast.Thetreatmentvolumeswere: primarytumorand
its local-regional lymphatic (perirectal, iliac and obturator)
drainages, with dosages from 45 to 50.40Gy administered
over25–28 days,from Monday toFriday. Afour-field
treat-ment was performed; the patient was placed in a prone
position,andthebelly-boardtechniquewasusedforbowel
loop removal and toxicity reduction. The patients treated
bythree-dimensionaltechniquewereevaluatedaccordingto
a dose/volume histogram, and the two-dimensional
treat-ment cases were evaluated with the use of treatment
curves.
Thechemotherapyprocedure consistedofaninfusional
chemotherapy,including5-fluorouracil(5-FU)andleucovorin
(Lv)inthefirstandlastfivedaysofradiotherapy;or, alterna-tively,bycontinuousoralingestionofcapecitabinethroughout radiotherapy.
Withrespectto whether ornot performing thesurgical
treatment,thetherapeuticconductwasdefinedbythose sur-geonsresponsibleforeachcase.
Patients undergoingsurgical treatment had their tumor
responsedefined byapathological analysis ofthesurgical
specimen,withtheresponsebeingclassifiedascompleteor
incomplete.
Bloodleukocyteprofileanalysis
Thenumber oftotalleukocytesand theexpressionof
lym-phocyteswereidentifiedbyreviewingaCBCperformedbefore
starting the treatment with radiotherapy and
chemother-apy.Therelationship betweenleukocytes andlymphocytes
wasobtained bythe followingcalculation: “total leukocyte
count divided by the total number of lymphocytes”
(neu-trophil/lymphocyteratio;N/L).
Dataprocedureandstatisticalanalysis
ThedatawerestoredinanExceldatabase(MicrosoftOffice
2003package)andanalyzedwiththeStatisticalPackagefor
theSocialSciences(SPSS)program,version17.0.
Ethicalaspects
ThestudywasapprovedbytheEthicsCommitteeofthe
Uni-versidadedaRegiãodeJoinville(UNIVILLE).Thestudybegan aftertheEthicsCommitteeapproval.
Results
Ofthe35patientsanalyzed,complete(clinicalorpathological) responseswereobservedin11cases(31.4%).Intwopatients,
weoptedforaschemeofobservationwithfrequentrevisions
(clinical response), while in nine cases surgical treatment
(pathologicalresponse)wasperformed.
Table1–Totalnumberofleukocytes.
Completeresponse N Mean SD p
Absent 24 7220 1999.2 0.838
Present 11 7390 2788.5
Patientswithcompleteresponsewhodidnotundergo surgery
These two patients were not submitted to surgical
treat-ment, as a complete tumor response, evaluated by digital
rectalexaminationandsigmoidoscopy,wasobserved.These
patientswerefollowedforamedianof24months.
Inthisgroup,themeanagewas82.5years;oneofthese patientswasaman.
In this observational period, there was no evidence of
tumorrecurrenceinbothpatients.
Operatedpatients
Of the 32 patients who underwent surgicalresection after
neoadjuvanttherapy,acompletepathologicalresponsewas
observedinnine (28%).Thisgrouphadamean ageof51.9
years;amongthosewhoachievedacompleteresponse,48.8
years;andforthosecasesoftumorpersistence,55years.
Non-operatedpatients
Onepatienthadthediseaseandcouldnotbeoperateddue
totheclinicalcondition.Thispatientwasconsideredasnot respondent,anddatawereincludedintheanalysis.
Bloodleukocyteprofileanalysis
Consideringthe totalnumber ofpatientswithcomplete or
incomplete response to neoadjuvant therapy, we observed
therespectivebloodleukocyteprofilesforeachgroup,as fol-lows.
Totalnumberofleukocytes
AsshowninTable1,theCBCobtainedpreviouslyto neoadju-vanttherapyshowedthattherewasnostatisticaldifferencein
totalnumberofleukocytesbetweenpatientswithandwithout
completeresponse.
Table2–Lymphocyteexpression.
Completeresponse N Mean SD p
Totallymphocytes
Absent 24 1960 608.3 0.474
Present 11 2103 338.9
%Lymphocytes
Absent 24 27 6.0 0.193
Table3–Neutrophil/lymphocyteratio.
Completeresponse N Mean SD p
N/Lratio
Absent 24 3.7 0.88 0.502
Present 11 3.5 1.21
Table4–Radiotherapydosage.
Completeresponse N Mean SD p
Absent 24 50 4.4 0.065
Present 11 47 2.6
Lymphocyteexpression
AsshowninTable2,theCBCobtainedpreviouslyto neoadju-vanttherapyshowedthattherewasnostatisticaldifference
regarding the absolute or relative number of lymphocytes
betweenpatientswithorwithoutcompleteresponse.
Neutrophil/lymphocyte(N/L)ratio
AsshowninTable3,theCBCobtainedpreviouslyto neoadju-vanttherapyshowedthattherewasnostatisticaldifference
regardingtheneutrophil/lymphocyteratiobetweenpatients
withorwithoutcompleteresponse.
Treatmentdose
Althoughtheradiotherapydosewasnotamongthevariables
initiallyestablishedforanalysisinthisstudy,the retrospec-tivelyobserveddifferencebetweenthemeansofcompleteand incompleteresponsegroups(50Gy×47Gy)ledustoassessits possibleprognosticvalue.AsshowninTable4,aborderline differenceforsignificance(p=0.06)wasobserved.
Excludedpatients
Aftertheselectionof96patients(35fromthecenterofJoinville and61fromthecenterofJaraguadoSul),61casesthatdidnot meetthecriteriaforinclusioninthestudywereexcluded.
Discussion
Tumorantiinflammatoryresponseplaysanimportantrolein
tumorevolutionresponse,includingcasesofcolorectal
can-cer.Severalstudieshavereportedthattumorinfiltrationby CD8+Tcells,whenactivated,showsprognosticvalueandgood correlationwithdiseasestaging,suggestingapossibleaction thatinducestumorcellapoptosis.Thislymphocyteactivation
canbedemonstratedbythelocalexpressionofelementssuch
asgranzimesB,CD69+orCD107+.AccordingtoKochetal.,an
increaseinthenumberofCD4+Tcellswasobservedinthe
mucousmembraneoftumorcells,comparedtonormaltissue
mucosa,suggestinganincreasedimmuneresponsein
colorec-taltumors.ThisprognosticvaluewasalsoobservedbyNaito
etal.;theseauthorsstudied131casesofcolorectalcancerand correlatedthelocationofCD8+cells,which,whenpresentin thevicinityofthecancercellnidus,showedbettersurvival.2–4
Inadditionoftumorresponse,thereiscompellingevidence
ofarelationshipbetweensystemicresponseandprognosis.
Several authorshaveshownthatthe numberofcirculating
lymphocytes,inparticularthroughaneutrophil/lymphocyte
(N/L)ratioanalysis,hasprognosticvalueincasesofcolorectal cancer.Liuetal.notedasignificantrelationshipbetweenN/L valueandsurvivalofpatientsundergoingtreatmentofrectal
cancer.Hungetal.analyzed1040patientswithcoloncancer
(stageII), ofwhom 785(75.5%)exhibited normalN/Lratios
and 255(24.5%)hadhigh N/Lratios.PatientswithhighN/L
hadlowerratesofoverallsurvivalandofdisease-freeinterval,
whencomparedtopatientswithnormalN/L.5
Chiangetal.evaluated3857patientswithcolorectal can-cerinstagesI–III,notingthatN/Lratios>3wereassociated withhighersensitivityandspecificityandlowersurvivalafter 5years,bothincoloncancer(66.3%×78.9%,p<0.001)andin colorectalcancer(60.5%×66.2%)comparedwithpatientswith
N/Lratiosbelowthisvalue.Furthermore,patientswithN/L
abovethislevelalsoexhibitedlargertumors(5cm)andamore advancedlocaldisease.6
Chua et al. analyzed 349 patients in two independent
cohorts treated withfirst-line palliative chemotherapy and
notedthatN/Lratioisapositiveprognosticfactor.7
Relationshipbetweensystemicinflammatoryresponse andresponsetoneoadjuvanttherapyinrectalcancer
Oncedetectingtheprognosticvalueofthesystemic
inflamma-toryresponse,someauthorshavesoughttodemonstrateits
possibleinfluenceonradiochemotherapyresultsperformed
in patients withrectal tumor. In a study that analyzed 73
patientsoperatedfortreatmentofrectalcancerafter
neoad-juvantradiochemotherapy,10(14%)achievedcompletetumor
response.Theauthorsobservedasignificantlyhigher periph-erallymphocytecount(p=0.02)andaneutrophil/lymphocyte ratiotendingtolowervalues(p=0.099).Basedonthese
find-ings,theauthorssuggestedthatthecompleteeradicationof
tumorcellsisdependentonanimmunereactionmediated
by lymphocytes, and that the maintenanceof the number
of circulating lymphocytes is an important factor in this
response to neoadjuvant therapy. The same authors, in a
subsequentstudy,analyzedbloodsamplestakenbeforeand
afterradiotherapyandconfirmedthatpatientswhoachieved
a complete response, with disappearance of tumor cells
(15/179),hadelevatedperipherallymphocytecountsandlow
neutrophil/lymphocyteratios.8
In a study that analyzed the tumor response to
radiochemotherapy with barium enema, the authors also
observed an association between this response and the
proportion oflymphocytes in relationto leukocytes. Other
authors related the results obtained in the treatment
of rectal cancer in 115 patients undergoing neoadjuvant
therapy with the use ofradiochemotherapy with the
neu-trophil/lymphocyteratio(N/L)obtainedfromperipheralblood, notingthatN/Lratios>5wereassociatedwithloweroverall survival,shortertimetolocalrecurrenceandlower
disease-free interval. Moreover, these patients showed a median
survivalof18.8monthscomparedwith54.4monthsforthose
Inaninterestingdevelopmentofthefindingsmentioned
above,astudywasconductedinwhichtheauthorssoughtto
correlatetumorresponsetoneoadjuvanttherapyinpatients
withrectal cancer with the apoptotic index obtained with
invitroirradiation oflymphocytesobtainedfrom peripheral
bloodofthesepatients.Asaresult,theseauthors achieved
asignificantcorrelationbetweenlymphocyteapoptosisand
histologicalregression,concluding thattumor
radiosensiti-vitycanbeestimatedfromtheapoptoticindexoflymphocytes invitro.10
Relationshipbetweenlymphocyticinfiltrationand responsetoneoadjuvanttherapyinrectalcancer
Withtheuseofimmunohistochemistry,Yasudaetal.analyzed
theleveloftumorinfiltration(LTI)byCD4+andCD8+Tcellsin biopsiesof48casesofadvancedcolorectalcancerbefore
com-pletionofneoadjuvantradiochemotherapy,andrelatedtheir
findingstothetumorreductionevaluatedbybariumenema
andbybiopsyafterneoadjuvanttherapy.Thenumbersofboth
CD4+and CD8+ lymphocytes inbiopsy samples previously
toradiochemotherapyshowedstrongcorrelationwithtumor
reduction,asassessedbybariumenema.Moreover,CD4+and
CD8+LTIdensitiesweresignificantlyassociatedwith histo-logicalgradeafterneoadjuvanttherapy.CD8+LTIdensitywas alsoanindependentprognosticfactorinachievingacomplete response.1
Inthe present study,the occurrenceofcomplete tumor
responseafterneoadjuvanttherapyforthetreatmentof
rec-talcancerwasobservedin31.4%ofcases,aresultconsistent
withthose observedin the literature. However,it was not
possibletodetectarelationshipbetweenthisresponseand
the analyzed elements in the leukocyte profile, including
the total numbers ofleukocytes and lymphocytes and the
neutrophil/lymphocyteratio.Webelievethatthesmall
num-berofcasesincludedinthis study mighthavecontributed
to this finding, due to factors inherent to a retrospective
study.
Ontheotherhand,thestrongtrendforasignificant
cor-relationbetweentheresponseandthedoseofradiotherapy
administeredcalledourattentiontotheneedfortakinga pos-siblygreatercontrol,inordertoreducethevariabilitybetween treatments,withtheestablishmentofmorerigidprotocolsfor optimizingtheneoadjuvanttherapyresults.
Conclusions
Inthis study, wecould notdemonstrate asignificant
rela-tionshipbetweencompletetumorresponseandneoadjuvant
therapyintheanalyzedleukocyteprofileparameters,
includ-ing a previous total number of leukocytes, absolute and
relativenumbersoflymphocytes,andneutrophil/lymphocyte
ratio.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Annex.
Table
of
pathological
response
grading.
Dworaketal.(14) Rodeietal.(17)
0.Noregression 0.Noregression
1.Predominantlytumorwith
significantfibrosisand/or vasculopathy
1.Regression<25%of
tumormass
2.Predominantlyfibrosiswith scatteredtumorcells(slightly recognizablehistologically)
2.Regressionof25–50%
oftumormass
3.Onlyscatteredtumorcellsin theareaoffibrosis
with/withoutacellularmucin
3.Regression>50%of
tumormass
4.Novitaltumorcellsdetectable 4.Completeregression
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