www.revportpneumol.org
ORIGINAL
ARTICLE
Bronchial-pulmonary
adenocarcinoma
subtyping
relates
with
different
molecular
pathways
Vítor
Sousa
a,b,∗,
Bruno
Bastos
a,
Maria
Silva
a,
Ana
Maria
Alarcão
a,
Lina
Carvalho
a,baIAP-FMUC-InstituteofPathology,FacultyofMedicine,UniversityofCoimbra,Coimbra,Portugal bPathologyInstitute,CoimbraUniversityHospital(HUC-CHUC),Coimbra,Portugal
Received30October2013;accepted30May2014 Availableonline6March2015
KEYWORDS Lung; Adenocarcinoma; APC; BCL2; CyclinD1; EGFR; ERCC1; HER2; LRP; MRP
Abstract Lungcancerisoneofthemostcommoncancersintheworldwithahighmortality rate.Weanalyzed45surgicalsamplesoftheadenocarcinoma,13withlymphnodemetastasis. APC,BCL2,chromograninA,CK5/6/18(LP34),CK20,CK7,cyclinD1,EGFR,ERCC1,HER2,Ki67, LRP,MRP,P53,RBandTTF1expressionswereevaluatedbyimmunohistochemistry(IHC).
HigherKi67,APC,ERCC1expressionsandlowerTTF1expressionwereidentifiedinadvanced stages(IIAandIIIA)ofadenocarcinomas,whichreflectamoreaggressive,lessdifferentiated, possiblyanon-TRUadenocarcinoma.
Acinar,micropapillaryandBA/lepidicadenocarcinomapatternswerethemostsimilar pat-terns andpapillary was the mostdifferentpattern followed by solid pattern, according to expression of these markers. Different adenocarcinoma patterns are engaged with differ-ent molecularpathways for carcinogenesis, basedonthe differencesofexpression. Acinar, BA/lepidicandmicropapillaryshowedhigherTTF1 expression(typeTRU),andpapillaryand solidpatternsrevealedlessTTF1expression,exhibitinganon-TRU/bronchialphenotype.Solid patternrevealedlowerHER2andhigherEGFRandERCC1(thiscomparedtopapillary) expres-sion;papillaryhigherHER2andlowerERCC1expressions;micropapillaryhigherRBexpression; andacinarlowerERCC1andhigherEGFRexpressions.CiclinD1seemstohavemoreimportance inacinarandBA/lepidicpatternsthaninmicropapillary.ERCC1proteinexpressionin micropap-illary,solidandBA/lepidicpatternsmayindicateDNArepairactivation.Inhibitionofapoptosis couldbeexplainedbyBCL2overexpression,presentinalladenocarcinomapatterns.MRP-1and LRPwereoverexpressedinallpatterns,whichmayhaveimplicationsfordrugresistance.
Furtherstudiesareneededtointerpretthesedataregardingtotherapyresponseinadvanced stagedbronchial-pulmonarycarcinomas.
©2013SociedadePortuguesadePneumologia.PublishedbyElsevierEspaña,S.L.U.Allrights reserved.
∗Correspondingauthor.
E-mailaddress:[email protected](V.Sousa).
http://dx.doi.org/10.1016/j.rppnen.2014.05.006
Introduction
Tobacco,environmentalandgeneticfactorsandseverallung diseasescontributetolungcancercarcinogenesis.Lung can-cerisoneofthemostcommoncancerdiagnosed,andhas alsothehighestmortalityrateduetotheadvancedstages at timeof diagnosis, andwhen theoptions for treatment havetobeunderstoodaspersonalizedtherapy.1---8
Bronchial-pulmonarycarcinomasconsistofsmallcelllung cancer(SCLC),squamouscellcarcinoma(SCC), adenocarci-noma,large cell carcinoma and sarcomatoid/pleomorphic carcinomas.8Adenocarcinomaisthemostcommontypein non-smokersanditsincidencehasincreasedinrecentyears, developing after terminal respiratory unit (TRU) but also recognized in respiratory epithelium.8---10 Adenocarcinoma purepatternsarerecognized[acinar,papillary, micropapil-lary,BA(bronchioloalveolar/lepidic/AIS-adenocarcinomain situ), solid with mucin production] but mixed type pre-dominates. Differences between the subtype/patterns of adenocarcinomashouldbe takeninto accounttoimprove diagnosis, prognosis and as an enhancement in therapy selection.8,10
Cytokeratin7(CK7),highweightmolecularcytokeratins (HWMC) like CK5/6, CK20, chromogranin A and TTF1 are currently usedin lung cancer diagnosis.11,12 Ki67 is a cell proliferationmarkerwithprognosticvalue.13---15 Inthe apo-ptosispathway, p53induces Baxtranscription andreduces bcl2.Bcl2/Baxratioisnegativeinlungcarcinomas.16 Inac-tivation of RB pathway is considered a requirement for lung carcinogenesis.16 Adenomatous polyposis coli (APC) gene is involved in the canonical Wnt/-catenin signal transduction. APC is mutated in liver, colorectal adeno-mas and lung tumors.17 Cyclins D (D1, D2, and D3) are activators of cyclin-dependent kinases cdk4 and cdk6, to translate growthsignals into cell cycle progression, lead-ingtoproliferation.18,19 Cyclin D1 overexpressionandpRB inactivationindicatesapoorprognosis.20
Bcl2 as an anti-apoptotic protein has a more impor-tant role in neuroendocrine (NE) lung cancer than in other bronchial-pulmonary carcinomas.21 Excision repair cross-complementing 1 (ERCC1) genetic polymor-phisms may affect patient’s response to platinum-based chemotherapy.22,23 However,anotherstudy concludedthat patients with a bronchial-pulmonary carcinoma with high ERCC1expression haveabetterprognosis thanthosewith low expression.24 Multidrug Resistance Protein 1 (MRP-1) and lung resistance-related protein (LRP) may confer resistancetocytotoxicandantiviraldrugs.25---31
EGFRandHER2playacentralroleintumorigenesis.32,33 Targetingthose receptors provides a uniqueapproach for treatingEGFR/HER2expressingcancers.34,35Themost com-mon alteration of HER2 is overexpression/amplification; mutationsarelessfrequent.33,35,36Theactivatedreceptors trigger Ras-Raf-MEK (mitogen-activated and extracellular signalregulatedkinase),ERK1andERK2(extracellular-signal regulatedkinase1and2)pathwaysleadingtocellgrowth, mTOR (mammalian target of rapamycin) pathway leading to protein synthesis, and PI3K-AKT (phosphatidylnositol-2 kinaseAkt)pathwaysustainingcellsurvival.36
The objectives of this work were to validate a small differentiation IHC panel, evaluate protein expression of
differentgenesaccordingtodifferentadenocarcinoma pat-terns, and correlate this expression with cell pathways of progression and drug resistance, in order to under-standbronchial-pulmonaryadenocarcinomacarcinogenesis. Anotherobjectivewastoverifypossibledifferencesofgene expressioninadenocarcinomatakingintoaccountvariables such asgender, age, smokingand differenttumor stages. The global intention of this project is to acquire knowl-edge whichcan be appliedin thediagnosis and prognosis evaluationofbronchial-pulmonaryadenocarcinomaandits subtypesinordertodefineindividualtreatment.
Materials
and
methods
Materials
Surgical samples ofbronchial-pulmonary adenocarcinomas (n=45)coveringallsubtypesrecognizedbyWHO/2004were randomlyselectedandstagedbythe2010TNMsystem;the ERS/ATS proposal was alsoapplied. At least twosections ofeachtumorandsectionsoflymphnodemetastasiswere selected(Table1).12,37
Methods
CK7, TTF1, CK20, CK 5/6/18 (LP34), chromogranin A, Ki67, P53, RB, BCL2, cyclin D1, APC, ERCC1, LRP, MRP-1, EGFR, and HER2 expressions were evaluatedby immuno-histochemistry(IHC).IHCwasperformedonformalin-fixed, paraffin-embedded tissue samples in automatic immunos-tainer (Autostainer 360 --- LabVision®). Streptavidin biotin protocolwasappliedaccordingtothemanufactures indica-tions for each antibody. Three-micrometer tissuesections wereplacedoncoatedslidesandallowedtodryovernight. After deparaffinization and rehydration, antigen retrieval was performed according to Table 2. Endogenous peroxi-dase activity was quenched using15min incubation in 3% dilutedhydrogenperoxide(H2O2).Forblockingnon-specific binding, Ultra V Block (Ultra Vision Kit®; TP-015-HL) was applied to the sections and then they were incubated at room temperature, with primary antibodies according to Table 2. After washing with phosphate-buffered saline (PBS),slideswereincubatedwithbiotin-labeledsecondary antibody (LabVision®) for 15min. Primary antibody bind-ing was localized in tissues using peroxidase-conjugated streptavidin(LabVision®)and3,3-diaminobenzidine tetrahy-drochloride(DAB)wasusedasthechromogen,accordingto manufacturer’sinstructions.Theslideswerecounterstained with hematoxylin, dehydrated and mounted. In parallel, knownpositive(Table2)andnegativecontrolswereused.
Three-micrometertissuesectionswereplacedoncoated slidesandallowedtodryovernight.IHCscoringwasapplied independently by two pathologists who registered the intensity of expression (1 --- low expression, 2 --- interme-diate expression and3--- high expression) andpercentage of stained cells. For statistical analyses we grouped the immunohistochemistryresultsin4categories.Foreachcase wemultipliedtheintensityofexpressionbythepercentage ofstainingcellsandweusedthisscorefortheformationof
Table1 Gender(M---male;F---female),age,smokingstatus,TNMclassification,stageandpatternspresent(A---acinar;B ---BA/lepidic;C---papillary;D---micropapillaryandE---solid).
Gender Age Tobacco TNM Stage Patterns
1 M 73 Non-smoker pT1aN0Mx IA A;B
2 F 71 Non-smoker pT1aN0Mx IA A;B
3 M 69 Smoker pT4N0Mx IIIA A;B;C
4 M 75 Non-smoker pT1aN2Mx IIIA A;E
5 F 53 Non-smoker pT1aN0Mx IA A;B
6 M 76 Ex-smoker pT2bN0Mx IIA A;B;E
7 F 62 Ex-smoker pT1aN2Mx IIIA A;B;E
8 F 74 Non-smoker pT1aN0Mx IA A;B;E
9 F 68 Non-smoker pT2aN0Mx IB A;D;E
10 F 50 Non-smoker pT1bN2Mx IIIA A;E
11 F 57 Smoker pT2aN0Mx IB A;E
12 M 51 Non-smoker pT1aN1Mx IIA A;B
13 F 75 Non-smoker pT2aN0Mx IB A;D;E 14 M 85 Ex-smoker pT1aN0Mx IA A;B 15 F 63 Smoker pT2aN0Mx IB A;B;D 16 F 55 Smoker pT1aN0Mx IA A;E 17 F 67 Smoker pT1aN0Mx IA A;B;D 18 M 48 Smoker pT1aN0Mx IA A;B;E
19 M 71 Non-smoker pT2aN1Mx IIA A;D
20 M 49 Smoker pT2aN0Mx IB A
21 F 80 Non-smoker pT1bN0Mx IA A;D
22 F 68 Non-smoker pT2aN1Mx IIA A;D
23 M 48 Non-smoker pT2bN0Mx IIA A;B 24 F 67 Non-smoker pT2aN0Mx IB A 25 M 76 Ex-smoker pT1bN0Mx IA A;B 26 F 56 Non-smoker pT1aN0Mx IA A;B;C;D 27 M 64 Smoker pT2bN0Mx IIA A;C 28 F 71 Non-smoker pT2aN0Mx IB A;B;E 29 F 50 Smoker pT2aN0Mx IB B;E 30 F 80 Non-smoker pT1aNxMx IA E 31 M 56 Smoker pT1aN0Mx IA A;B 32 F 56 Non-smoker pT1aN0Mx IA A;B 33 F 54 Non-smoker pT1aN0Mx IA B
34 M 67 Smoker pT2aN1Mx IIA A;E
35 F 62 Non-smoker pT1bN1Mx IIA A;C;D
36 F 68 Non-smoker pT1bN0Mx IA A;B
37 F 44 Non-smoker pT2bN2Mx IIIA A;E
38 F 52 Smoker pT3N1Mx IIIA A;B;D
39 M 45 Smoker pT2aN1Mx IIA A;C
40 M 61 Smoker pT2aN0Mx IB A
41 F 74 Non-smoker pT2aN0Mx IB A;D
42 F 74 Non-smoker pT2aN0Mx IB A;B
43 M 73 Non-smoker pT2aN1Mx IIA A;B
44 F 60 Non-smoker pT1bN0Mx IA A;B;D 45 F 79 Non-smoker pT2aN0Mx IB A;B;D
eachcategory(category(−)0---10;(+)11---100;(++)101---200; (+++)201---300)(Table3)
The non-parametric Wilcoxon-Mann---Whitney test was usedtoanalyzethedifferencesbetweencategories. Hier-archicalclusteringandPrincipalComponentAnalysis(PCA) wasmade tovalidaterelationships betweenpatterns. We usedthe software STASTISTICA 7. P valuesless than 0.05 wereconsideredsignificant.
Results
Predominantlymixed-typeadenocarcinomas,40threeacinar adenocarcinomas, one solid mucin producing adenocarci-noma and one bronchioloalveolar (BA)/lepidic mucinous adenocarcinoma/mucinous adenocarcinoma in situ were studiedtovalidatethedifferentpatterns. Forty-twocases (93.3%) with acinar pattern (being the most prevalent),
Table2 Antibodiesappliedantigenretrieval,dilutionandincubationtimeandstainingpatterns.RT---roomtemperature. Primaryantibody Manufacture Clone Positivecontrol Antigen
retrieval
Dilutionand incubationtime
Staining pattern APC Novocastra EMM43 Colon PTmodule,
EDTA
1:40,60 Membraneand cytoplasmic Bcl2 Novocastra 3.1 Tonsil PTmodule,
EDTA
1:150,60 Membraneand cytoplasmic ChromograninA DAKO DAK-A3 Carcinoidtumor PTmodule,
citrate
1:200,30 Cytoplasmic CK(5/6/18) Novocastra LP34 Lung PronaseE(10,
RT)
1:100,60 Cytoplasmic CK20 DAKO KS20.8 Colon PronaseE(10,
RT)
1:50,30 Cytoplasmic CK7 DAKO KS20.8 Lung PronaseE(10,
RT)
1:50,30 Cytoplasmic CyclinD1 Novocastra P2D11F11 Breastcarcinoma PTmodule,
EDTA
1:30,60 Nuclear EGFR Zymed 31G7 Respiratoryepithelia PronaseE(10,
RT)
1:20,30 Membraneand cytoplasmic ERCC1 Thermo 8F1 Tonsil PTmodule,
EDTA
1:100,60 Nuclear HER2 DAKO Polyclonal Breastcarcinoma PTmodule,
citrate
1:200,30 Membrane Ki67 DAKO MIB-1 Smallcellcarcinoma PTmodule,
citrate
1:50,30 Nuclearexcept mitoticcells LRP Novocastra 9D6 Ileon PTmodule,
EDTA
1:300,30 Cytoplasmic MRP-1 Novocastra 33A6 Colon PTmodule,
citrate
1:50,60 Cytoplasmic P53 DAKO DO-7 Skin PTmodule,
citrate
1:50,30 Nuclearand cytoplasmic RB Novocastra 13A10 Tonsil PTmodule,
EDTA
1:50,60 Nuclear TTF1 DAKO 8G7G3/1 Smallcellcarcinoma PTmodule,
EDTA
1:100,60 Nuclear
26 (57.8%) withBA/lepidic pattern, 15 (33.3%) withsolid pattern, 5 (11.1%) with papillary and 15 (33.3%) with micropapillarypatternwereregistered.
Thirteen cases (28.9%) showed lymph node metastasis asfollows:acinar patternwaspresentin 13cases(100%); solidand micropapillarypatterns werepresent in 3cases each (23%);and papillarypattern in 2 cases(15.4%). Two metastasiswithsolidpatternbelongedtoprimary adenocar-cinomaswhichharbored,respectively,acinarandBA/lepidic andacinar,BA/lepidicandmicropapillarypatterns.
Seventeen cases (37.8%) were diagnosed in men and twenty-eight cases (62.2%) in women. Age at diagnosis ranged from 44 to 85 years with a median of 67 years. Fourteencases(31.1%)werediagnosedinsmokers(8males and6 females), four(8.9%)in ex-smokers (3males and1 female)andtwenty-seven(60%)inneversmokers(6males and21females).Femalepatientsweremorefrequently non-smokersthanmen(p=0.0251).
Seventeen cases (37.8%) were diagnosed as stage IA (5 males/12 females), twelve (26.7%) as IB (2 males/10 females), ten (22.2%) as IIA (8 males/2 females) and six (13.3%) as stage IIIA (2 males/4 females). Females were
diagnosedmorefrequentlyinlowerstageswhencompared to men (p=0.029). There were no differences between stagesaccordingtosmokinghabits.
Alladenocarcinomas(n=45)expressedCK7.No expres-sionwasobservedforCK20,HWMCorchromograninA.TTF1 wasexpressedinthegreatmajorityoflung adenocarcino-mas,withstatisticalsignificanthigherexpressioninacinar pattern(p=0.0094),micropapillarypattern(p=0.012)and BA/lepidicpatterns(p=0.00046).Theexpressionwas signif-icantlyhigherinBA/lepidicpatternwhencomparedtosolid pattern(p=0.021).
NuclearexpressionofP53wasstatisticallyhigherinall patternswhencomparedtonormaltissue(p<0.05),without differencesbetweenpatterns.
RBexpressionwashigherinmicropapillarypatternwhen comparedtonormaltissue(p=0.011). Theother patterns showednodifferencesfrombasalexpressioninnormal tis-sue.
Therewerenosignificant differences betweenprimary adenocarcinomasandtheirmetastasis,exceptforcyclinD1 whose expressionwashigherinthemetastasis when com-paredtotheprimaryneoplasia(p=0.0409).
Table3 TTF1,KI67,P53,RB,APC,BCL2,CiclinD1,ERCC1,MRP1,LRP,HER2andEGFRexpressionaccordingtoadenocarcinoma pattern.A---acinar;B---BA/lepidic;C---papillary;D---micropapillaryandE---solidpatterns.Expressioncategories:0=(−)=0---10%; 1=(+)=11---100%;2=(++)=101---200%;3=(+++)=201---300%.
APCexpressionwashigherinallpatternswhencompared to normal tissues (p<0.05), with nodifferences between patterns.
BCL2 expression was higher in all patterns studied thannormaladjacenttissue(p<0.05),withoutdifferences betweenpatterns.
CyclinD1expressiondifferedbetweennormaltissueand solid(p=0.0002)andBA/lepidic(p=0.002)patterns,being higherinthesepatternsthaninthenormaltissue.
EGFRproteinexpressionwashigherinacinar(p=0.033) and solid (p=0.012) patterns when compared to normal tissues, concerningmembrane expression only. Ifwe take intoaccountcytoplasmicexpressiontheoverallexpression wassignificantly higherin allpatternswithout differences betweenthem.
ForHER2we observedsignificantlyhigherexpressionin all patternscomparedto normaltissue (p<0.05). Expres-sion is also higher in acinar, papillary, micropapillary and BA/lepidicpatternscomparedtosolidpattern(p<0.05).
ERCC1 expression was lower in acinar (p=0.0016) and papillary (p=0.019) patterns compared to normal tissue. Expressionintheotherpatternsrevealednodifferencesto basalexpressioninnormaltissue.Papillarypatternhas sig-nificantlessexpressionthansolid(p<0.05)andBA/lepidic patterns(p<0.05).
Ki67 expression was higher in all adenocarcinoma pat-ternswhencomparedtonormallungtissue(p<0.05).There werenodifferencesbetweenthepatterns.
LRP expression was different (higher) between nor-mal and the different patterns of the adenocarcinomas (p<0.05).
MRP-1expressionwashigherinallpatternscomparedto normallung tissue, without differences between patterns (p<0.05).
Some immunohistochemical results are illustrated in
Fig.1.
Theanalysisofthedendrogramclearlyshowedthat ade-nocarcinoma is genetically different from normal tissue (Fig.2).Concerningthepatterns,BA/lepidicandacinarare themoresimilarandpapillarythemostdistinct or genet-icallyseparatedpattern. ByPCAanalysis(Fig.3)wewere abletoidentifythreeclusters:thefirstcomposedof papil-larypatternadenocarcinomas;thesecondcorrespondingto solid;andthethird tothegroup ofacinar,BA/lepidicand micropapillarypatterns.
Looking at gender, we have identified higher expres-sionofERCC1infemalepatientswhencomparedtomales (p=0.0008). We did notfindany differences according to age. HER2 expression was higher in adenocarcinomas of non-smokers or ex-smokers (p=0.0384). Ki67 expression
Figure1 (A)Expression(++)ofAPCinBApattern,200×;(B)Expression(+++)ofBCL2inpapillarypattern,200×;(C)BApattern withCyclinD1expression,400×;(D)CyclinD1expressioninsolidpattern,200×;(E)EGFRexpression(+++)inacinarpattern,200×; (F)EGFRexpression(+++)insolidpattern,200×;(G)ERCC1expression(++)inBApattern,200×;(H)ERCC1expression(++)insolid pattern,200×;(I)HER2expression(++)inacinarpattern,400×;(J)Ki67expression(+++)inlymphnodemetastasis,200×;(L)Ki67 expression(+++) inacinarpattern,400×;(M)LRPexpression(+++)inacinarpattern,400×;(N)MRP-1expression(+++)insolid pattern,200x;(O)RBexpression(++)inpapillarypattern,200×;and(P)P53expression(+++)inmicropapillarypattern,200×.
Tree diagram for 6 variables single linkage euclidean distances Normal Acinar BAC Micropapillary Solid Papillary Linkage distance 3.5 3.0 2.5 2.0 1.5 1.0 0.5
Figure2 Dendrogramforthehierarchicalclusteringanalysis oftranscriptsaccordingtotheirexpressionvalues,usingSingle linkagealgorithm, Euclideandistances, targeting thevarious patternsandnormaltissue.
washigherin adenocarcinomasof smokersandex-smoker patients(p=0.0034andp=0.0064).
TTF1,Ki67,APC,RB,P53,BCL2,ERCC1andEGFRshowed significantlydifferentexpressionsaccordingtothestageof the tumor. TTF1 expression showed differences between stage IAand IIIA, being higherin stage IA (p=0.02).Ki67 was higher in IIIA compared to IA and IIA (p=0.02 and p=0.02). APC expression was higher in IIA and IIIA com-pared to IA (p=0.065 and p=0.025). RB expression was higherin stageIB tumorscomparedtostage IA(p=0.04). P53expressionwashigherinIIA,IBandIIIAcomparedtoIA (p=0.01;p=0.03;p=0.01).BCL2expressionwashigherfor IB compared to IA (p=0.039). ERCC1 washigher in stage IA compared to IIA (p=0.02) and in IIIA compared to IIA
BAC
Projection of the variables on the factor-plane (1 x 2)
Papillary Acinar Solid 1.0 0.5 0.0 –0.5 –0.0 –1.0 –0.5 0.0 0.5 1.0 Factor 1: 87.53% Factor 2: 6.36% Active Cropapillary
Figure3 Thecorrelationofthepatternswiththeadvanced PCAfactoranalysis.
(p=0.01).EGFRexpressionwaslowerinstageIBcompared toIA(p=0.032).
To sum up, almost all patterns showed differences of expressionin relation tonormaltissues withsome excep-tionsforcyclinD1(papillaryandmicropapillarypatterns), for EGFR membrane staining (papillary and micropapil-lary patterns), for ERCC1 (in micropapillary, solid and BA/lepidic patterns), for RB (in acinar, papillary and solid patterns) and for TTF1 (in papillary and solid pat-terns).
SolidpatterndemonstratedlowerHER2expression com-pared to acinar, papillary, micropapillary and BA/lepidic patterns.PapillarypatternhadlowerERCC1expressionthan solidandBA/lepidic patterns.Solid patternshowedlower TTF1expressionthanBA/lepidicpattern.
Discussion
Non-BA/lepidic adenocarcinomas are most often periph-eral tumors with different growth patterns considered mixed type according to WHO classification, with at least two individual patterns; solid or acinar patterns are the most frequent in the central tumoral bulk and BA/lepidic at the periphery. Acinar has been described as the most common pure subtype and the most com-mon in the mixed type. Papillary pattern was present in 10% of the adenocarcinomas, often difficult to differen-tiate from BA/lepidic, which has important therapeutic strategies’ implications.9 Micropapillary pattern is cha-racterized by small papillary tufts growing from alveolar septa or floating within alveolar spaces lacking fibrovas-cular cores and is usually observed with papillary and mixed patterns; they are more aggressive due to their increased capacity for metastasize, indicating unfavor-able prognosis.9 Solid pattern with mucin production has been reportedin 15% of mixed-type adenocarcinomas.9,10 BA/lepidic pattern shows tumor cell growth along alveo-lar septa and aerogeneous spread through the lung. It is frequently diagnosed in former and non-smoking women. BA/lepidic is classified as non-mucinous, mucinous or mixed.9,10,38
Markersofdifferentiation
CK20,HWMCand chromograninAwerenegativeinallour samplesandCK7andTTF1werepositive,whichmeans mor-phologyalmostalwayssustainsbronchial-pulmonary adeno-carcinomas’ diagnosis when mucin is not present. These resultscorroboratedthosethathavebeendescribedinthe literature.CK7is positivein90---100% andCK20ispositive inonly7---10%ofprimarylungadenocarcinomas.39 Chromo-graninAisnotexpressedinanordinaryadenocarcinoma.40 TTF1 is a useful marker for lung adenocarcinoma and is alsoexpressedinthyroid normaltissueandtumors.22 The use of TTF1, as a differentiation marker, is useful not onlyfor diagnosticpurposebut especiallytodifferentiate betweendifferenthistologicaltypes ofbronchial andTRU adenocarcinomas.
Geneticexpressionineachpatternof bronchial-pulmonaryadenocarcinoma
Acinarpattern
Starting withmembrane proteins that play a central role in the control of cellular proliferation, differentiation, apoptosisand angiogenesis, EGFR and HER2 had a higher expressioninacinarpatternthaninnormalepithelium/lung cells,highlighting theimportanceofAKT/mTOR,PI3Kand MAPKpathways,essentialforoncogenictransformationand tumormaintenance.41Blockageofbothresultsinapoptosis andtumorshrinkage.42,43 AKT,byinhibitingGSK-3,changes canonicalWntpathway,releasing-catenin,andleadingto angiogenesisandproliferation.44
TTF1positivitywasstatisticallyhigherinthe adenocar-cinomas(withacinar pattern)comparedtonormal tissue. WhilethemajorityoftheadenocarcinomasexpressesTTF1, thismarkerismisunderstood,becauseitspositivityis associ-atedinverselywithproliferationbutitiscorrelatedwithP53 expressionand HER2/neuinvolved in lung cancer.45 TTF1, NKX2-8andPAX9 areveryimportant ininitiationand pro-gressionoflungcancer.ItissaidthatMAPKinase,JAK/STAT andWntareimportantinactivationofTTF1andNKX8, con-nectedwithEGFRandHer2.46
The expressionofcyclin D1washigherin tumoralthan in normal tissueand RB expression hadno statistical dif-ference when compared to normal tissue. This fact may indicateatendencytoproliferate,becauseoncecyclinD1is inhighconcentration,itcouldphosphorylatemoreRB pro-teinandreleaseE2F,involvedintheregulationofgeneswith implicationsin cell cycle progression(G1 toS phase).43,47 One of the genes that correlate with E2F is Ki67, still a goodmarkerfor proliferative index.48 Weobtained statis-ticalsignificanthigherexpressionofKi67inallpatternsof adenocarcinoma,indicating active proliferative activity.49 Another overexpressed gene in this pattern was P53 and some studies indicate that ‘‘a low level of p53 leads to transient growth arrest and cell survival, whereas a high levelpromotesirreversibleapoptosis’’,andP53levelsmay haveoscillations,andithastobeexploredasatargetfor therapy.50ERCC1isdownregulatedinacinarpattern,maybe indicatingthatDNArepairpathwayisnotactivatedinthe acinarpattern.ERCC1isassociatedwithradioresistance,so itisimportantfortreatmentchoice.51HighBCL2expression mayfavorcellcyclearrest.
LRPandMRP1werealsooverexpressedinoursamples,as hasbeendescribedinlungcancer.52,53Asmentionedthese genescanleadtochemotherapyresistance.Thisfactshould betaken into consideration when assessing prognosisand treatmentissues.
APC was also overexpressed in the acinar pattern, and being involved in -catenin degradation is important in progression, with GSK-3 and Axin downregulating -catenin(leading to-catenin degradation).54 Ourfindings alsosuggestthat-cateninmaybedownregulated,already describedin lung cancer, whichis relatedtocell invasion and metastasis.55 -Catenin is connected to cytoskeleton and cadherins and regulates E- or N-cadherin-mediated celladhesion.Notchactivation(thatinactivates-catenin) may repress E-cadherin, leading to tumor growth and metastasis.56 So,overexpressionofAPC leadsto-catenin
degradation that weakens cell adhesion promoting tumor growthandmetastasis.
In relation to the acinar pattern the genes analyzed expressions could lead to cell proliferation and survival, angiogenesisandmetastasis.
Papillarypattern
This patternshowedsomedifferencescomparedtoacinar pattern.Therewerenodifferencesofexpressionforcyclin D1andTTF1,comparedwithnormaltissue.SincecyclinD1 level is not upregulatedas well as RB gene, the concen-tration of active E2F must be regular. However Ki67 was overexpressed, incontrasttoprevious findings.21 Soother pathways could explain cellular proliferation. There was higherexpressionofEGFRbutjustincytosolcompartment; membraneEGFRhadthesamelevelofexpressionasnormal tissue,sowecanconcludethatactivityofEGFRisnormal. Thus,allpathwaysthatthisgeneregulatesarefunctioning normallyor if activated,thisactivation is not induced by EGFR.41,57,58
Papillarypatternproliferationpropertiescouldinpartbe explainedby APC upregulation when compared tonormal tissue.
Micropapillarypattern
Micropapillary pattern is characterized by small papillary tuftsandisconsideredanaggressiveadenocarcinoma,more than papillary pattern.59---61 There were three differences withpapillarypatternsconcerningexpressionofERCC1,RB and TTF1.RB expression wasdownregulatedin micropap-illary, being almost non-existent. Some authors said that downregulationofRBleadstotheprogressionofcellcycle.62 MicropapillarypatternhadabasallevelofERCC1,this indi-catingthatDNArepairmayoccurwithNERactivation.TTF1 wasoverexpressedinthispatternand,accordingwithwhat hasbeensaidbefore,thisfactindicateslungcancer progres-sionand developmentafterthe terminal respiratoryunit. Theseconsiderationsareconsistentwithourresultsbecause downregulationofRBandTTF1overexpressionare responsi-bleforcellcycleprogressionandbasallevelofERCC1allows DNArepair.Thislastconsiderationisveryimportanttokeep inmindintreatinglungcancer.
Solidpattern
Genetic expression in this pattern is identical to acinar pattern with only two differences for ERCC1 and TTF1 expressions.Theseshowednodifferencescomparedto nor-maltissueinsolidpattern,whereasinacinarpatternthere was a higher expression for TTF1 and downregulation for ERCC1.AlowerTTF1expressionreflectsalower differenti-ationofthispatternandeventuallymeansamoreaggressive behavior since in general less differentiated tumors are moreaggressive.HigherEGFRexpressioncouldbethereflex of EGFR mutations and/or overexpression activating sev-eralpathways involved in cellcycle control, proliferation andevenmetastization.EGFRoverexpressionisalso associ-atedwithpoorprognosis.33,63SolidpatternhasmoreERCC1 expression than acinar and this aspect is very important becauseERCC1repairsDNAandconfersresistancetosome drugs.51
BA/lepidicpattern
BApattern,calledlepidicinATS/ERSproposedclassification can bedifficult todistinguish morphologically from papil-larypattern.12CyclinD1hadhigherexpressioninBA/lepidic pattern and a basal level in papillary pattern. This fact andknowingthatRBwasnotoverexpressed,enhancesthe idea thatthis pathway leads to cellprogression. As TTF1 is related with tumor progression and is overexpressed, unlikeinpapillarypattern,andERCC1hasabasallevelin BA/lepidic pattern and is downregulatedin papillary pat-tern,thismayexplainchemicalresistance.
By evaluating the expression of these 13 genes, BA/lepidic pattern has more cell progression pathways activated than papillary pattern. This finding proves that they are biologically different. The differences could be explainedbytheorigininTRUwiththedifferentactivated pathways, withprobable implicationin the prognosis and treatment,knowingthatdifferentpathwaysaretargetsfor therapeuticapproach.
Metastasisanalysis
Metastasisformationisacomplexprocesswithseveral path-waysactivatedleadingtolossofcellularadhesion,increased motility andinvasiveness.55 Acinarpattern metastasis had cyclin D1 over-expression, statistically different between primaryandmetastatictumor.Thisgeneisinvolvedincell progressionthatmightbehelpingcellsurvivalincirculation andcolonization.55
Gender,age,smokingandtumorstaging
ERCC1expressionwashigherinfemales,correlatinginsome studiestoabetterprognosis.64 HoweverERCC1mayconfer resistancetocisplatin.65
Lung adenocarcinoma is more frequently diagnosed in patients over 60 years andit is infrequentbefore age 40 andgenerallymoreaggressive.66Alloursamplesweretaken frompatientsolderthantheageof40.Therewereno dif-ferencein theexpressionof themarkersstudiedbetween thegroupunderandover60years.
Concerningsmokinghistory,HER2expressionwashigher in non-smokers than in ex-smokers. Ki67 expression was higher in tumorsof smokers and ex-smoker patients than in non-smokers, indicating a higher proliferative rate and moreaggressive/mitoticallyactivetumors.
Womendiagnosedwithadenocarcinomaweremore fre-quentlynon-smokers,reflectingtheculturaltobaccohabits andsomeofthoseadenocarcinomasbelongingtothegroup ofadenocarcinomasdiagnosedinwomen,non-smokersand young thatcould harborEGFR mutations.67 Adenocarcino-mas diagnosed in women patients were more frequently diagnosed at stages IA and IB reflecting somehow a less aggressivebehaviordemonstratedalsobyalower prolifera-tiveindexinthesestages.Tobaccohabitscouldalsoexplain theactivationofdifferentpathwaysandgeneticalterations in these cases. Our results also demonstrated a relation betweenhigherKi67expressionandsmokinghabits.
Manygeneproductshavetheirexpressionalteredwhen correlating withstage, such as APC, BCL2, EGFR, ERCC1, Ki67,P53,RB andTTF1,showingthatgeneexpressionis a
dynamic and unforeseen process.From stage IA toIB we observed lowerBcl2 and EGFR expressions and RB higher expression.FromstageIBtoIIAweobservedhigherBcl2and lowerERCC1 expressions. IIIA stage showedhigher ERCC1 and Ki67 expressions. From IA and IB to IIA we observed alsohigher APC and Bcl2 expressions. In advanced stages weobservedespeciallyhigherKi67,APCandERCC1 expres-sionsandlowerTTF1expression,thelastreflectingalower differentiationandevenanon-TRUadenocarcinomawitha moreaggressivebehavior. Ki67higherexpressioninhigher stages wasan expectedresult asit reflects higher prolif-erationindexandthus aggressivebehavior. APCmutations weredescribedinseveraltumors.17Takingintoaccountour results,itseemsthatAPCgeneticandpathwayschangesare importanttoconsiderinadvancedstagesof adenocarcino-mas.
Oneof the most important molecule in this study was P53becauseitwashigherexpressedinallpatternsandis involvedinseveralpathwaysandmechanismsinnormalcell cycle, carcinogenesis and DNA repair with obvious thera-peutic implications. Some authors relate P53with MRP1, becausetheirparallel increaseisfrequent. Itwas demon-stratedthatP53wild-typesuppressedMRP1promoter.These hypothesesarecorroboratedbyourdata,becausewealways obtained high expression of MRP1 and P53. According to theseauthorsP53wasmutatedinallpatterns.68
RBexpressionwaslowerinmicropapillarypattern,which alsoexplaineda moreaggressive phenotypebecausefree E2Factivatestranscriptionofgenesrelatedwithtumor pro-gression.
TTF1 was connected with P53 and HER2 in lung can-cer and some authors relate this protein with initiation and progression in lung carcinogenesis.19,23 The relation betweenTTF1 andHER2 wasobserved between solidand BA/lepidic patterns, because both gene expression was higherinBA/lepidicpatternthaninsolid.
Conclusions
Femaleadenocarcinomapatientsweremorefrequently non-smokersanddiagnosedinearlierstages,withhigherERCC1 expressioninvolvedinDNArepair.
Advanced stages (IIA and IIIA) of adenocarcinomas showedhigherKi67,APC,ERCC1expressionsandlowerTTF1 expressionreflectingamoreaggressive,mitoticallyactive and less differentiated adenocarcinoma and eventually a non-TRUadenocarcinoma.
Therewasgenerallyhigherexpressionoftheproductsof genesstudiedintheadenocarcinomascomparedtonormal adjacentcells reinforcingtheirimportanceinlung adeno-carcinomacarcinogenesis.
There were two specific gene expressions with differ-encesbetweenpatterns,HER2andTTF1thatinterferewith gene transcription. Papillary and solid patterns revealed lessTTF1expression,identicaltonormaltissueexhibiting anon-TRU/bronchial phenotype. At the other end acinar, BA/lepidicandmicropapillaryshowedhigherTTF1 expres-sion.SolidpatternrevealedalsolowerHER2andhigherEGFR andERCC1(thiscomparedtopapillary)expression.Papillary showedhigherHER2andlowerERCC1expressions. Adeno-carcinomasshowed higher TTF1 expression (type TRU) in
acinar,BA/lepidicandmicropapillarypatterns, micropapil-larywithhigherRBexpression,acinarwithlowerERCC1and higherEGFR.CiclinD1 seemstohave moreimportancein acinarandBA/lepidicpatternsthaninmicropapillary.
ERCC1 protein expression in micropapillary, solid and BA/lepidicpatternsindicatesDNArepairandinacinarand papillary patterns there was lower expression. BCL2 was overexpressedinallpatterns,suggestingthatthereis inhi-bitionofapoptosis.MRP-1andLRPwereoverexpressedinall patternsanditisimportanttofurtheranalyzetheseproteins forabetterunderstandingoftheresponsetotherapy.
By hierarchical clustering test we highlight papillary pattern as the most different pattern followed by solid pattern; acinar and BA/lepidic patterns are most alike. UsingPCA analysiswe realizedthat acinar,micropapillary and BA/lepidic patterns are the most alike, so we made threeclustersof patterns: papillary,solidand micropapil-lary/acinar/lepidic(BA).
Furthersstudies areneeded inordertointerpretthese resultsregardingtherapeuticresponsein advancedstaged bronchial-pulmonarycarcinomas.
Ethical
disclosures
Protection of human and animal subjects.The authors declarethatnoexperimentswereperformedonhumansor animalsforthisstudy.
Confidentialityofdata.Theauthorsdeclarethattheyhave followedtheprotocolsoftheirworkcenteronthe publica-tionofpatientdata.
Right to privacy and informed consent.The authors declarethatnopatientdataappearinthisarticle.
Funding
FundedbyagrantfromCIMAGO,FacultyofMedicine, Uni-versityofCoimbra,Portugal.
Conflicts
of
interest
Theauthorsdeclarethattheyhavenoconflictsofinterest.
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