Journal
of
Coloproctology
w w w . j c o l . o r g . b r
Original
Article
Clinical-pathological
and
p53
protein
expression
study
in
dysplasia
associated
with
ulcerative
colitis
夽
,
夽夽
Antônio
Baldin
Júnior
a,b,∗,
José
Ederaldo
Queiroz
Telles
c,d,e,
Renato
de
Araújo
Bonardi
a,f,
Heda
Maria
Barska
dos
Santos
Amarante
a,g,
Rosimeri
Kuhl
Svoboda
Baldin
a,d,eaUniversidadeFederaldoParaná(UFPR),Curitiba,PR,Brazil
bColoproctologyUnit,HospitaldeClínicas,UniversidadeFederaldoParaná(UFPR),Curitiba,PR,Brazil
cUniversidadeFederaldeSãoPaulo(UNIFESP),SãoPaulo,SP,Brazil
dHospitaldeClínicas,UniversidadeFederaldoParaná(UFPR),Curitiba,PR,Brazil
eDepartmentofClinicalPathology,UniversidadeFederaldoParaná(UFPR),Curitiba,PR,Brazil
fDepartmentofSurgery,UniversidadeFederaldoParaná(UFPR),Curitiba,PR,Brazil
gDepartmentofInternalMedicine,UniversidadeFederaldoParaná(UFPR),Curitiba,PR,Brazil
a
r
t
i
c
l
e
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n
f
o
Articlehistory:
Received19November2013
Accepted4February2014 Availableonline16June2014
Keywords:
Ulcerativerectocolitis Dysplasia
Proteinp53
a
b
s
t
r
a
c
t
Background:The associationbetweenulcerativecolitis andadenocarcinomadetermined
strategiesforpatientfollow-upandearlydetectionofdysplasticandneoplasticlesions.
Aims:Toanalyze theincidenceofdysplasia inpatientswithulcerative colitis,
compar-ingclinicaldataofpatientswithandwithoutdysplasiaandcheckimmunohistochemical expressionofp53proteinindysplasias.
Materialsandmethods:Weanalyzedbiopsysamplesandclinicaldataof124patientswith
ulcerativecolitisatHospitaldeClínicas,FederalUniversityofParaná,Curitiba,Brazil.
Results:Dysplasiaincidencewaslow(9.67%)andallcaseswithlow-gradedysplasia.Patients
clinicaldatacomparisonwithandwithoutdysplasiadidnotshowsignificantstatistical dif-ferenceswithregardtotherace,ageatthestartofthedisease,ageatlastbiopsy,durationand anatomicextentofulcerativecolitis.Significantdifferencewasfoundbetweenmalesand femaleswithpredominanceofmales(58.34%)fordysplasia.Seventeenthbiopsysamplesof 12patientswithdysplasia,5(29.4%)werep53positive.
Conclusions:Fromtheseresultsitisconcludedthattheincidenceofdysplasiawaslow,higher
inmalesandtherewaspositivityofp53proteinindysplasia.
©2014SociedadeBrasileiradeColoproctologia.PublishedbyElsevierEditoraLtda.All rightsreserved.
夽
StudyconductedattheDepartmentofPathology,HospitaldeClínicas,UniversidadeFederaldoParaná,Curitiba,PR,Brazil. 夽夽
PresentedasaposterattheXXVBrazilianCongressofPathology,Natal,RN,2005.
∗ Correspondingauthor.
E-mail:antoniobaldinjr@ufpr.br,ksbaldin@ig.com.br(A.BaldinJúnior). http://dx.doi.org/10.1016/j.jcol.2014.02.009
Estudo
clínico-patológico
e
da
expressão
da
proteína
p53
nas
displasias
associadas
à
retocolite
ulcerativa
Palavras-chave: Retocoliteulcerative Displasia
Proteínap53
r
e
s
u
m
o
Racional: Aassociac¸ãoentreretocoliteulcerativaeadenocarcinomadeterminouestratégias
paraseguimentodospacientesedetecc¸ãoprecocedaslesõesdisplásicaseneoplásicas.
Objetivos: Analisar a incidência de displasia nos pacientes com retocolite ulcerativa,
comparardadosclínicosdospacientescomesemdisplasiaeverificaraexpressão imunois-toquímicadaproteínap53nasdisplasias.
MaterialeMétodos:Foramestudadososexamesanatomopatológicosedadosclínicosde124
pacientescomesemdisplasia,portadoresderetocoliteulcerativanoHospitaldeClínicas daUniversidadeFederaldoParaná.
Resultados: Aincidênciadedisplasiafoide9,67%etodososcasosforamdedisplasiade
baixograu.Nacomparac¸ãodosdadosclínicosdospacientescomesemdisplasianãohouve diferenc¸aestatisticamentesignificativacomrelac¸ãoàcor,idadenoiníciodadoenc¸a,idade naúltimabiópsia,extensãodadoenc¸aetempodeevoluc¸ãodadoenc¸a.Houvediferenc¸a estatísticacompredomíniodepacientesdosexomasculino(58,34%)emrelac¸ãoaofeminino paradisplasia.Dos17examesavaliadosde12pacientescomdisplasia,em5exames(29,4%) aexpressãodaproteínap53foipositiva.
Conclusões: Dessesresultadosconclui-sequeaincidênciadedisplasiafoibaixa,maiorno
sexomasculinoehouvepositividadedaproteínap53nasdisplasias.
©2014SociedadeBrasileiradeColoproctologia.PublicadoporElsevierEditoraLtda. Todososdireitosreservados.
Introduction
Ulcerativecolitis(UC)fallswithinthegroupofchronic
inflam-matory bowel diseases ofunknown cause.With outbreaks
ofremissionandexacerbation,UChasanincidenceof3–20
newcasesper year per100,000 inhabitants.1 UC withover
8years ofevolutionrepresentsanimportantriskfactor for
thedevelopmentofdysplasiaandsubsequentdevelopmentof
adenocarcinoma.2,3Theincreasedreplacement(turnover)of
intestinalepithelialcellsdamagedbychronicinflammation
isconsidered a risk factor for the development of
dyspla-siaandadenocarcinomainpatientswithlongprogression.4
The risk of developing colorectal cancer varies from 5.5%
to13.5%,andriskfactorsincludetheextentofdiseaseand theprogressiontime.5Monitoringandfollow-upofpatients
are performed bymeans of colonoscopies and serial
biop-sies,but these cover less than 0.05% ofthe colic surface.6 Geneticalterationshavebeenstudiedtocharacterize dysplas-ticlesionsand foranearlydetectionofcarcinogenesis.5 Of
all the markersstudied, the expressionofp53protein has
demonstratedsignificantcorrelationwithdurationofdisease, asariskfactorofdevelopingcolorectalcancerassociatedwith UC.7
Althoughtheexpressionofp53isalateeventincolorectal carcinogenesis,itisconsideredanearlyeventintheonsetof dysplasiaassociatedwithUC.8Theonsetofexpressionofp53, byanabnormalprotein,inthenucleusofepithelialcellsofthe intestinalmucosaoccursevenwithoutthepresenceof dyspla-siaandprecedesthedevelopmentofdysplasiaandcolorectal cancer.9
Material
A retrospective study was performed since 2004 in 124
patients with clinical and endoscopic diagnosis ofUC
fol-lowedattheinflammatoryboweldiseasesoutpatientservice,
Hospital de Clinicas, Universidade Federal do Paraná, and
who underwent pathological examination of the colonic
mucosa. This study was approved by the Research Ethics
Committee,HospitaldeClinicas,withCEP/HC
732.151/2003-10protocol.InthedatabaseoftheDepartmentofPathology
ofthesamehospital,allpatientsandtheirrespectiveexams
werereviewed.Aclinicaldatasurveywasconductedforage,
gender,race,progressiontimeofthediseaseuntilthetimeof
examination,andextentofdisease.Pathologicallabworkup
included endoscopic biopsies and surgical specimens. The
pathologicalreportsinwhichwerementioned“indefinitefor
dysplasia(IND)”,“presenceofdysplasia”or“adenocarcinoma” wereselected.
Incaseswithdysplasiaandintwocasesofcolic
adeno-carcinoma,immunohistochemicalreactionsfornuclearp53
proteinwereperformed.
Method
This study included 124 patients with ulcerative colitis,
followed-up bybiopsies, andthe timeelapsedbetweenthe
siteofdisease.Theexplanatoryvariablesweredichotomized bytesting thenullhypothesisofdistributionsof dysplasia-freetime(timeelapsedfromdiseaseprogressiontodiagnosis ofdysplasia,ortothelastbiopsy)equalinbothclassifications versusthealternativehypothesisofdifferentdistributionsof dysplasia-freetime.Thestatisticaltestusedwasconsidered theCox-Manteltest.Theevaluationoftheprogressiontimeof
thegroupswithandwithoutdysplasiawasperformedusing
thenon-parametricMann–Whitneytest.pvalues<0.05were consideredstatisticallysignificant.
Theindefinitecasesfordysplasiaandthosewithdysplasia werereviewedwiththeuseofadiscriminantclassificationto characterizedysplasiaasatypiaor repair.10 Afterreviewing
the pathological examinationreports, the cases were
clas-sifiedas“withdysplasia”, “withoutdysplasia”or“indefinite
fordysplasia”.Theparaffin-impregnatedmaterialwascutin
4-mmthick-sections.Preparationofimmunohistochemistry
slidesandtheirdeparaffinizationandhydrationwithxylene
anddecreasingconcentrationsofethanolwereperformed,as
wellasantigenretrievalwithcitratebuffer.Mousemonoclonal
antibodyanti-p53protein inpreviouslyparaffined material
wasused.ThisamonoclonalantibodyofimmunoglobulinG2
class,that bindstoboththe wild-typeand tothe mutated
protein. The antibody was used at a dilution of 1:100. A
reaction using the streptavidin–biotin-peroxidase complex,
using colon adenocarcinoma p53-positive as positive
con-trol,wasperformed. Acountingofbrown-stainednucleiby
meansofanimageanalyzer(Image-ProPlus® –Theproven
solutionTM Version 4.5.1.23for Windows98/NT/Me/2000/XP
Copyright©1993–2002MediaCyberneticsInc.)wasperformed. Positivecellswithnucleistronglybrown-stainedwerecounted bytheprogrambycolordifferenceversusnucleiinblue (neg-ative cells). Lesions with less than 10% positive cells were
negativeforp53; between1%and 25%,positive+, between
26%and50%,positive++,greaterthan50%,positive+++.11
Results
Thirty-eight(30.65%) male and 86 (69.35%) female patients
wererecruited.Nine(7.26%)wereblackormestizopatients
and115(92.74%)were Caucasians.Clinicaldata(age,ageat
onsetofdisease,diseaseduration,andnumberofbiopsies)
areshowninTable1.
Regardingtheextentofdisease,78(62.90%)patientshad
onlyinvolvementofleftcolonand46(37.09%)exhibited pan-colitis.
Ofthe124patients, in12 (9.67%)wefound20
patholog-icalexaminationsdiagnosedwithlow-gradedysplasia(LGD)
orIND,17withLGD(Fig.1)and3withIND.Noexamination
withhigh-gradedysplasiawasfound.
Twofemalepatientswithadenocarcinomahadconcurrent
dysplasia.Onepatientdevelopedadenocarcinomainhepatic
colonicflexure,after8yearsofdiseaseprogression,withstage
T2N0 M0; Astler-Coller B1;DukesA. Another patient
pre-sentedwithrectaladenocarcinomawithsignetringcellsareas
after12yearsofdiseaseprogression,withstageT2N1M0;
CollerAstler-C1-2;DukesC.
Ofthe17testsevaluated(from12patientswithdysplasia), in5(29.4%)theresultwasp53-positive(Fig.2)andin12(70.6%)
Fig.1–Areaofdysplasia.Note:Dysplasticnucleidark blue-stained.HE400×.
Source:DepartmentofPathology,UFPR.
wasp53-negative. Anexaminationwithpositivity+(24%of
cells),1withpositivity++(39%ofcells)and3withpositivity +++(>50%ofcells).Twocasesofdysplasiawereassociated withamass(DALM),withapositivity+++.Thefemalepatient witharectaltumorwasp53-positive+++andtheotherpatient hadhertumorinrightcolonflexurewasp53-negative.
In the statistical analysis with dichotomized variables,
dysplasia-freetimedidnotcorrelatewithrace(p=0.9467)nor withtheextentofthedisease(p=0.1551).Theagebeforeor after15yearsatdiseaseonsetandpatientage<40yearsor≥40
yearsatthelastbiopsydidnotcorrelatewithdysplasia-free time(p=0.8882andp=0.7920respectively).InFig.3,itappears
that maleshad higherincidenceofdysplasiainrelationto
dysplasia-freetime(p=0.0242).Theprogressionofdiseasein
patients with and without dysplasia showed no difference
(p=0.8055).
Fig.2–Dysplasiap53-positive.Note:Dysplasticnuclei brown-stainedbrownbeforemarkingforcellcount. Immunohistochemistryforp53,400×.
Table1–Clinicaldata.
Variable n Minimum Maximum Median Mean Standard
deviation
Ageatlastbiopsy 124 10.00 81.00 39.00 39.71 13.48
Ageatthebeginningofthedisease 124 3.00 79.00 32.50 33.90 13.56
Diseasedurationatlastbiopsy 124 1.00 36.00 5.00 6.81 5.97
Nr.ofbiopsies 124 1.00 10.00 3.00 3.60 2.17
Source:TheAuthor.
Discussion
Themedicalattentionisfocusedonthemonitoringofpatients withUC,and thedysplasiafoundinpathological examina-tionsbecamethemostsignificantpredictivefactorofcancer.12 Onestudyfound13.1%ofdysplasiain590patientsundergoing
proctocolectomyanddeterminedthatthepositivepredictive
valueforcoloniccancerofapreoperativefindingofdysplasia ofanygradeis50%.13
Low-grade dysplasiacan bedetectedin17% ofpatients
duringfollow-up with endoscopicbiopsies.14 In this study,
LGD was detectedin 12 patients (9.67%). Higher incidence
ofdysplasiainpatientswithconcomitantadenocarcinomais
related;inoursample,twopatientswhohadadenocarcinoma
alsohadLGD.13
Someauthorsfoundafive-foldhigherincidenceof
can-cerinpatientswithpancolitisthaninpatientswithleftside colitis.15Anotherstudyfound18patientswithLGD;17ofthem hadpancolitis(94%).16Inthisstudy,disagreeingwiththe
find-ingsoftheauthorsabovementioned,ofthe12patientswith
dysplasia,seven(58.30%)hadpancolitisandfive(41.70%)had leftcolitis.Theextentofdiseasedidnotinfluencethe occur-renceofdysplasiawithstatisticalsignificance(p=0.1551).
Thetimeofdiseaseprogressioniscitedasariskfactorfor colorectalcancer.15,17Inoursampletherewasnoagreement withthisobservation(Table2).Thecomparisonofgroupswith
andwithoutdysplasiashowednosignificantdifferencewith
regardtotimeofdiseaseprogression(p=0.8055).
Male Female
Dysplasia-free time Male x female
Complete Censored
Ulcerative rectocolitis duration (years)
Cumulative population of cases without dysplasia
0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00
40 35 30 25 20 15 10 5 0
P=0.242 (cox-mantel)
Fig.3–Dysplasia-freetimeandgender.
Source:Theauthor.
Table2–Frequencydistributionofdiseasedurationat lastbiopsy.
Follow-up(years) Withoutdysplasia Withdysplasia
Frequency Percentage Frequency Percentage
<5 50 44.64 4 33.33
5–9.9 35 31.25 5 41.67
10–14.9 16 14.29 3 25.00
15–19.9 7 6.25 – –
≥20 4 3.57 – –
Total 112 100.00 12 100.00
Source:TheAuthor.
Inthisstudytherewasapredominanceofwhitepatients, whichisalsoobservedintheUnitedStates.18Inthisseries,
thegroupwithdysplasiahad11whitepatientsandoneblack
patient.However,whenanalyzingtheinfluenceofraceand
freedysplasia-freetime,therewasnosignificantdifference (p=0.9467).
Theexpressionofthenuclearproteinp53hasbeenstudied asamarkerfordisplasia.7Althoughthemutationofthep53 geneisconsideredtobealateeventinthecarcinogenesisof sporadiccoloniccancers,inthecancerassociatedwithcolitis, themutationappearsasanearlierevent.7,9 Inthissample, fiveexamswithLGDwerep53-positive(29.4%).Thisfindingis consistentwiththeliterature,whereLGDpositivityindexesof 25–50%arefound.9,19
In thisstudy,the tests withDALMshowed dense
stain-ing(+++)forp53.ThepresenceofaDALMinjurycorrelatesin
43%ofcaseswithaconcurrentfindingofadenocarcinoma.2
A strong expression of p53 is found in DALM and not in
adenoma.20
In the United States, women are more affected by UC
than men.18Accordingly,wefoundafemalepredominance
inpatientswithulcerativecolitis(69.35%).TheEuropean
epi-demiologicalstudywasdiscordantfrom theUSAstudy and
foundhigher incidenceinmeninthe agegroupsabove 35
years.21Inoursample,wefoundmoremeninthegroupwith
dysplasia, and the statistical analysis showed a significant
difference(p=0.0242).Somestudieshavefoundahigher pro-portionofcaseswithdysplasiaandcolorectalcancerinmales, buttherewasnostatisticalassessmentofthesignificanceof thisdifferenceinrelationtogender.22–24
Conclusions
Theincidenceofdysplasiawaslower thanthatinthe
dysplasia.Theincidenceofdysplasiawassignificantlyhigher inmales.Theexpressionofp53proteinindysplasiaswas con-sistentwiththeliterature.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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