Anais
Brasileiros
de
Dermatologia
www.anaisdedermatologia.org.brINVESTIGATION
Contribution
to
characterization
of
skin
field
cancerization
activity:
morphometric,
chromatin
texture,
proliferation,
and
apoptosis
aspects
夽,夽夽
Anna
Carolina
Miola
a,b,∗,
Mariana
Anteghini
Castilho
c,
Juliano
Vilaverde
Schmitt
a,
Mariangela
Esther
Alencar
Marques
d,
Helio
Amante
Miot
aaDepartmentofDermatologyandRadiotherapy,FaculdadedeMedicinadeBotucatu,UniversidadeEstadualPaulista,Botucatu,
SP,Brazil
bDepartmentofDermatology,InstitutoLaurodeSouzaLima,Bauru,SP,Brazil
cDisciplineofRadiologyandDiagnosticImaging,FaculdadedeMedicinadeBotucatu,UniversidadeEstadualPaulista,Botucatu,
SP,Brazil
dDepartmentofPathology,FaculdadedeMedicinadeBotucatu,UniversidadeEstadualPaulista,Botucatu,SP,Brazil
Received26January2019;accepted22March2019 Availableonline25October2019
KEYWORDS Carcinoma,squamous cell; Keratosis,actinic; Skinneoplasms Abstract
Background: Askinfieldcancerizationisacutaneousareawithsubclinicalchangesresultant
fromchronicsunexposure,withahigherpredispositiontodevelopmentofpre-neoplasticand neoplasticlesions. Sofar,therearenowell-definedobjectiveparametersthatcanindicate theirdegreeofactivity.
Objectives: Todescribeandcomparemorphometricaspectsandexpressionoffactorsrelated
toapoptosisandcellproliferationinactinickeratosis(AK),inbothphotoexposedand photo-protectedepidermis.
Methods: Across-sectionalstudyofpatients withactinickeratosisintheforearms, biopsied
attwopoints:theactinickeratosisandtheaxillaryregion.Thebiopsiesoftheactinic kerato-sis,perilesionalarea,andaxillawereevaluatedthroughkeratinocyteintraepithelialneoplasia (KIN),and immunohistochemistryofp53,survivin, andKi67.Nuclearmorphometry ofbasal layercellswasperformedthroughdigitalimageanalysis:entropy,area,perimeter,Ra,fractal dimension,circularity,colorintensity,andlargestdiameter.
夽 Howto citethisarticle: Miola AC,Castilho MA,Schmitt JV, Marques MEA, MiotHA. Contribution to characterizationof skinfield
cancerizationactivity:morphometric,chromatintexture,proliferation,andapoptosisaspects.AnBrasDermatol.2019;94:698---703.
夽夽StudyconductedattheFaculdadedeMedicinadeBotucatu,UniversidadeEstadualPaulista,Botucatu,SP,Brazil.
∗Correspondingauthor.
E-mail:annafmrp@yahoo.com.br(A.C.Miola).
https://doi.org/10.1016/j.abd.2019.03.003
0365-0596/©2019SociedadeBrasileiradeDermatologia.PublishedbyElsevierEspa˜na,S.L.U.ThisisanopenaccessarticleundertheCC
Results: Therewere13patientsincludedand38actinickeratosisbiopsied.Inmorphometry, 1039nucleiwereanalyzed,ofwhich228representedaxillaryskin,396demonstratedactinic keratosis, and415 represented the perilesional area to the actinic keratosis. There was a significantdifference(p<0.05)inallvariablestestedfor thetopographiesevaluated.A sig-nificantcorrelationwasidentifiedbetweennucellarmorphometricelements,KIN,proliferation markers,andapoptosis.Jointpatternsofp53,Ki67,andKINdiscriminatedthetopographies sampled.
Studylimitations: Thiswasacross-sectionalstudywithasmallnumberofpatients.
Conclusions: Therearepatternsofproliferation,resistancetoapoptosis,anddifferentcellular
morphometricsbetweenphotoprotectedskinandphotoexposedskin.Thejointexpressionof p53,Ki67,andKINcancharacterizeskinfieldcancerizationactivity.
©2019SociedadeBrasileira deDermatologia.PublishedbyElsevierEspa˜na,S.L.U.Thisisan openaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).
Introduction
Theconceptoffieldcancerizationwasproposedby
Slaugh-terin1953 andcomprisesaseeminglynormal integument
area,butwithsubclinicalandmultifocalalterations,
com-posedofcellsgeneticallyalteredasaresultofchronicsun
exposure.1,2 The conceptof skin fieldcancerization (SFC)
suggeststhattheclinicallynormalskinadjacenttoactinic
keratoses(AKs)is thefocusofclonalexpansionof
geneti-callyalteredcells,which wouldexplaintheoccurrenceof
newAKs or other cutaneous neoplasms in thesame area,
inadditiontolocalrecurrenceoftumorsconsidered
com-pletelyexcisedbyhistopathologicalanalysis.3
Recently,SFChasbeenstudiedduetoitsclinical
impor-tance;itsstabilizationcanpreventtheappearanceof
neo-plasias,andtherecurrenceorevolutionofexistinglesions.
Currently, AKs, which are pre-neoplastic epithelial
lesions,areconsideredmarkers ofSFCactivity.4 However,
sofar,therearenowell-definedobjectiveparametersthat
canindicatetheirdegreeofactivity.
Determiningthe extent andintensity ofthe activityof
an SFC is important in order to create diverse and
ade-quatetreatmentandpreventionstrategiesforeachtypeof
patient,aswellastoevaluatetheresponseandprognosis
aftertheestablishmentofappropriatetherapies.5Survivin,
p53,andKi67 arenuclearmarkerspresent inproliferative
cells, and areexpressed in neoplastic and pre-neoplastic
lesions,whichmayaidinthecharacterizationofanactive
SFC.6
Synthesisofp53inducesDNArepairandapoptosis.
Muta-tions in p53 are the most common genetic alterations in
human neoplasms.7 Recently, theanalysis of exons in the
p53geneintumorsof theaerodigestivetracthas
demon-strateda probablemolecularmethodfor thedetectionof
thecancerizationfield.8 Inaddition,mutations inp53can
befoundinAKsandsquamouscellcarcinomas(SCCs)inmost
cases,andinhigherconcentrationsinSCC,whereaspatients
withoutsuspectedcancerlesionsdonotexpressp53
muta-tions,suggestingthatmutationsinp53maybeinvolvedin
theconversionofAKtoSCC,and,consequently,mayindicate
activityinthefieldofcutaneouscancer.9
TheKi67antigenisamarkerofcellproliferation10,11
cor-relatedwithtumorgrowthandhigherriskofmetastasis.12,13
ThenuclearexpressionofKi67hasbeenpreviouslystudied
in cutaneous neoplasms and is evidenced in AK, Bowen’s
disease,basalcellcarcinoma(BCC),andSCC.14,15 Todate,
thereare no studies describing the expression of Ki67 in
SFCactivity.
Survivin is a protein expressed by proliferative
ker-atinocytesandcanbefoundinthecytoplasmornucleus.16
It acts in regulation of the cell cycle and in control of
apoptosis.Itsfunctionvariesaccordingtotheintracellular
location17:whilenuclearexpressionismoreassociatedwith
celldivisionandisessentialformitosis,thepresenceof
sur-vivininthecytoplasmisconsideredcytoprotective.18Ahigh
levelofnuclearsurvivinispredictiveofgreatermalignancy
orworseprognosiswhenanalyzedinepithelialtumors.19---22
Moreover, the location of nuclear survivin in the
epider-mislayersalsoinfluencesandindicatesprognosis:whilethe
presenceofsurvivininthebasallayercanbefoundin
unal-teredepithelium,theexpression ofnuclearsurvivininthe
suprabasallayersindicatesruptureofepidermalcell
home-ostasisandconsequently,mitoticactivity,whichmayaidin
thecharacterizationofSFC.23
Nuclearmorphologicalanalysiscan providecluesabout
cellular physiology and contribute to the diagnostic and
prognostic evaluation of neoplastic lesions. In addition,
changes in nuclear chromatin architecture may indicate
intensemitoticactivityandcorrelate withneoplastic
pro-liferation rates. Thus, morphometry and nuclear texture
characteristics have been studied asprognosticfactors in
manyneoplasms.24---27Investigationsonnuclear
morphome-tryand chromatinheterogeneity have been performed on
BCCs; however, there are no studies characterizing such
parametersinSFCandcorrelatingthemwithfieldactivity.28
Theuseofmorphometricandimmunohistochemical
tech-niques,together with clinical and histopathological data,
canprovideinformationonthebiologicalbehaviorand
con-tributetotheevaluationoftreatmenteffectiveness.
Methods
Across-sectionalstudy wasconductedwithadultpatients
ofbothgenders,withAKintheforearms,biopsiedat two
points:theskincomprisinganAKandanotherpointofthe
axillary skin. This studywas approvedby the institution’s
ethics committee and all patients enrolled signed an
informedconsent.
The biopsies of the AK, the perilesional area, and
(HE)usingthe keratinocyteintraepithelial neoplasia(KIN)
method.KINclassificationisawayofclassifyingthe
inten-sityofkeratinocyteatypiabyitspresenceintheepidermal
levels,sothatKINIpresentsfocalatypiainthelowerthird
of the keratinocytes; KIN II shows depolarization in the
prickly-granulosalayers;andKINIIIshowsdepolarizationin
allepidermallayers.29 Forpurposes ofstatistical analysis,
normalaxillaryskinwasconsideredasKIN0.
For immunohistochemistry, the percentage of cells
stainedbysurvivin,p53,andKi67inthehotspotsoftheAK,
perilesional,andaxillaryareaswereanalyzed.
Theslideswerephotographedinhighresolution(400×)
usingaCoolscopeII microscope (NikonInstruments Inc.
---Tokyo,Japan,2009),withabout30nuclei/field.Thebasal
layer epidermal nuclei were sliced manually, and their
images were transformed to eight bits and normalized.
ImageJ software was used (Fig. 1). The resulting images
weresubmittedto theextraction of entropy,color
inten-sity,fractaldimension,Ra,circularity,perimeter,andarea
indices.
Regarding thestatistical analysis, datawere compared
betweenthetopographiesbytheJonckheere---Terpstratest.
Normality wasestimated by the Shapiro---Wilk test.30 The
correlationsbetweentheindicatorswereestimatedbythe
Spearmancoefficientandpresentedasathermalmap.31The
behaviorsofthevariableswerecomparedbymultiple
cor-respondenceanalysisandarrangedbyaperceptualmap.32
Valuesofp<0.05wereconsideredsignificant.
Results
Thisstudyincluded13patients:fourwomenandninemen,
from37to88yearsold,inwhom38AKsweresampled.
Inthemorphometry,1039nucleiwereanalyzed,ofwhich
228representedaxillarywholeskin,396werefromAKs,and
Standardized photography 8-bit transformation and image normalization Nuclei selection
Figure 1 Representative scheme ofimage preparationand nucleiselection.
415werefromtheperilesionalareasoftheAKs.Themean
morphometricvaluesareshowninTable1.
Therewasasignificant difference(p<0.05)in the
val-uesofphotoprotectedandphotoexposedskin inalltested
variables(p53,Ki67),asobservedinTable2.Inaddition,KIN
wasdirectlyrelatedtophotoexposure,tendingtowardzero
inthephotoprotectedskin(Fig.2).
Correlation between morphometric, proliferation, and
apoptosisindicators wasidentified(Fig. 3).The elements
Table1 Valuesofthevariablesaccordingtothelocationoftheskinsample.Allvariablesresultedinp<0.05.
Nuclearparametersa Axilla Perilesional AK
Area 1,278.30(1,128.0---1,266.62) 1,481.57(1,312.56---2,070.25) 1,823.58(420.54---2,295.45) Perimeter 140.98(135.64---145.06) 157.52(148.81---209.99) 178.52(152.27---213.58) Circulation 0.81(0.75---0.84) 0.74(0.71---0.77) 0.73(0.69---0.78) Largerdiameter 51.69(48.96---52.45) 56.62(54.15---69.30) 52.11(54.17---74.93) Medianofcolor 33.94(30.22---35.78) 39.19(31.59---43.38) 43.64(36.12---50.24) Entropy 5.30(5.17---5.32) 5.26(5.18---5.35) 5.34(5.26---5.54) Ra 1,346.55(988.80---2,026.91) 2,181.88(1,601.59---7,777.78) 3,579.69(1,744.43---8,115.73) Fractaldimension 2.45(2.44---2.45) 2.46(2.45---2.48) 2.47(2.45---2.50) aMedian(p25---75).
Table2 Percentage ofcellsexpressingsurvivin,p53, andKi67intheAK,photoexposed skin,andphotoprotectedskin.All variablesresultedinp<0.05.
Markersa Axilla Perilesional AK
Ki67 18.68(14.29---26.62) 23.98(17.59---29.46) 42.07(35.03---51.61)
Suprabasalsurvivin 11.58(6.71---15.50) 18.57(10.06---28.43) 38.16(23.22---46.41)
p53 13.81(5.38---20.12) 23.88(16.83---37.78) 65.35(54.64---79.61)
20 15 10 5 0 Axilla Periles QA Topography Counting 12 1 13 8 18 19 0 1 2 3 KIN
Figure2 Distributionofthekeratinocyteintraepithelial neo-plasia(KIN)evaluationaccordingtotopography.
thatmostcorrelatedwithKINwerep53andKi67.Therewas
anintensecorrelationbetweenthemorphometricelements.
Noneofthevariablestestedwashighlightedasan
indi-vidualmarkerofSFCactivity;however,KIN,p53,andKi67
togethercould characterize the behavior of topographies
(Fig.4). Therewasgreatspatialproximity ofAKwithKIN
3,andthehighestvaluesofKi67andp53;theexposedskin
hadcloseproximitytoKIN1,andintermediatevaluesofthe
othermarkers.However,theaxillaryepidermishadgreater
proximitytoKIN0.
Discussion
The SFC, in this group of samples, was characterized by
increasedepithelial proliferation, increased expression of
apoptosis-related factors,and alterations in nuclear
mor-phology,in agreementwiththeliterature.9,11 There wasa
progressiveexpression ofthesevariablesinprotected skin
inrelation tothesun-exposed skin andAKs, showingthat
chronic exposure to ultraviolet radiation promotes tissue
changesintheSFC,withcorrelationbetweentheexpression
ofthesefactorsandthedegreeofhistologicaldysplasiaof
theepithelium. CIRCULAR 1 0.5 -0.5 -1 0 RA FRACTAL AREA PERIMETER DIAMETER ENTROPY MEDIAN SURV P53 Ki67 KIN CIRCULAR RA FRA C T A L
AREA PERIMETER DIAMETER ENTR
OPY
MEDIAN SUR
V
P53 Ki67 KIN
Figure3 Thermalmapofthecorrelations(Spearman)betweenthemorphometricandapoptosis-relatedindicatorsand prolifer-ation.Allcorrelationsresultedinp<0.05.
2 1 0 -1 -2 -2 -1 0 Dimension 2 (iner tia = 49%) Dimension 1 (inertia = 78%) 60-93 3 37-93 QA 2 23-58 1 8-25 0-23 26-36 Axilla Topography Ki67 KIN P53 Periles 1 2
Figure4 Perceptualmapofthedistributionofp53,Ki67,and keratinocyteintraepithelialneoplasia(KIN)accordingto topog-raphy.
TheassociationsofepitheliawiththeexpressionofKIN,
p53,andKi67changedproportionallyaccordingtosun
expo-sure, which suggests them as strong candidates for the
characterizationofthe SFC,andindicates thatthe
analy-sisoftheiractivitycanbeusedintherapeuticclinicaltrials
andin thedevelopment ofmeasures aimed at preventing
thedevelopmentofskincancers.
Therewasalsoanincreaseintheexpressionofsuprabasal
survivin,witharelationdirectlyproportionaltotheincrease
ofsolar exposureand presenceof AA,asexpected inthe
literature23; however, its correlation with Ki67 and p53,
which are well established markers,11---14 was less intense
(Graph 2). In addition, further correlation studies are
neededbetweentheexpressionof suprabasalsurvivinand
SFCactivitysothatthereisahigherlevelofevidencefor
thesefindings.
Alterationsinnuclearmorphometryandchromatin
tex-ture are used in pathology as criteria for differentiation
betweenhealthytissuesandneoplasias.24,31Inaddition,
tis-suesunder stress, such as ultraviolet radiation, may also
present nuclear alterations, usually related to the
inten-sityofmetabolicactivity.24,28 Inthisstudy,phenotypically,
thegenomic damagetotheSFCmanifested aschangesin
theshapeofthenucleus andheterogeneity ofchromatin,
correlatedamongthemselvesandwithprogressivechanges
accordingtophotoexposureandthepresenceofAKs,
sup-portingtheevidenceforgenomicinstabilityandsuggesting
thatthesealterationsoccurinasimultaneousintranuclear
manner.
Themainlimitationsofthestudyarethesmallnumberof
participants,althoughtheyallowedasufficientnumberof
lesionsfortheanalysis.Asthestudyistransversal,although
AKandSFCareunstableentities,alongitudinaldesigncould
providemoreconsistentelementsoftheiractivitylinkedto
theoncologicalprognosis.33,34
The histologicalcharacterizationof SFCshouldprovide
additional contributions to the AK count, quality of life
scales, and clinical severity scores4,35,36 in understanding
theunderlyingphenomenaofSFC.Thesefindingsarebeing
testedinclinicaltrialsofSFCtreatmentssuchas
photody-namictherapy,37 ingenolmebutate,and5-fluorouracil,and
accordingtoascaleof severityof AKsindevelopmentfor
thepresentauthors’SFCstudygroup.
Conclusion
Morphometric and chromatin texture variables showed
differences incomparison of photoexposedand
photopro-tected skin. KIN, p53,and Ki67 --- together--- allowed the
characterizationofSFC.
Financial
support
Nonedeclared.
Author’s
contribution
Anna Carolina Miola: Approval of the final version of the
manuscript; elaboration and writing of the manuscript;
obtaining, analyzing and interpreting the data; critical
reviewoftheliterature.
MarianaAnteghiniCastilho:Approvalofthefinalversion
of the manuscript;conception andplanning of the study;
obtaining,analyzingandinterpretingthedata.
JulianoVilaverdeSchmitt:Approvalof thefinalversion
ofthemanuscript;obtaining,analyzingandinterpretingthe
data;effectiveparticipationinresearchorientation;
intel-lectual participation in propaedeutic and/or therapeutic
conduct of the casesstudied; criticalreview ofthe
liter-ature;criticalreviewofthemanuscript.
MariangelaEstherAlencarMarques:Approvalofthefinal
versionofthemanuscript;criticalreviewofthemanuscript.
Helio Amante Miot: Statistical analysis; approval of
the final version of the manuscript; effective
participa-tion in research orientation; intellectual participation in
propaedeuticand/ortherapeuticconductofthecases
stud-ied;criticalreviewoftheliterature.
Conflicts
of
interest
Nonedeclared.
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