PTEN as a predictive marker of response to conservative treatment in endometrial hyperplasia and early endometrial cancer. A systematic review and meta-analysis

Review

article

PTEN

as

a

predictive

marker

of

response

to

conservative

treatment

in

endometrial

hyperplasia

and

early

endometrial

cancer.

A

systematic

review

and

meta-analysis

Antonio

Travaglino

,

Antonio

Raffone

*

,

Gabriele

Saccone

,

Luigi

Insabato

,

Antonio

Mollo

,

Giuseppe

De

Placido

,

Fulvio

Zullo

a

AnatomicPathologyUnit,DepartmentofAdvancedBiomedicalSciences,SchoolofMedicine,UniversityofNaplesFedericoII,Naples,Italy

b

GynecologyandObstetricsUnit,DepartmentofNeuroscience,ReproductiveSciencesandDentistry,SchoolofMedicine,UniversityofNaplesFedericoII, Naples,Italy

ARTICLE INFO Articlehistory: Received26July2018

Receivedinrevisedform6October2018 Accepted8October2018 Availableonlinexxx Keywords: Immunohistochemistry Fertility-sparing LNG-IUD

Endometrialintraepithelialneoplasia Progestin

Progesteroneresistance Phosphataseandtensinhomolog

ABSTRACT

Objective:Severalmarkershavebeenstudiedtopredicttheresponsivenessofendometrialhyperplasia

(EH)andearlyendometrialcancer(EEC)toprogestintherapy.PTENhasplayedamajorroleinthisfield,

althoughitspredictivesignificanceisstillundefined.WeaimedtoassessiflossofPTENexpressionon

pre-treatmentendometrialspecimenmaybeapredictivemarkersofresponsetoprogestinsinEHand

EEC.

StudyDesign:MEDLINE,EMBASE,WebofSciences,Scopus,ClinicalTrial.gov,OVIDandCochraneLibrary

weresearchedforrelevantarticlesfromtheinceptiontoMay2018.AllstudiesassessingPTENexpression

aspredictivemarkerinEHandEECtreatedwithprogestinwereincluded.Relativerisk(RR)fortherapy

failurewascalculatedwith95%confidenceinterval(CI)andasignificantp-value<0.05,withasubgroup

analysisbasedonthehistologiccategory(EECorEH)andtheadministrationrouteofprogestin(oralor

intrauterine).

Results:Sevencohortstudiesassessing376patientswereincluded. PTENlosswas notsignificantly

associatedwiththeoutcomeoftherapyintheoverallanalysis(RR=1.24,95%CI,0.88–1.76,p=0.21),

in+thesubgroupsofEEC(RR=0.89,0.32–2.49,p=0.83),EH(RR=1.30,0.90–1.87p=0.16),oralprogestin

(RR=1.250.88–1.79,p=0.22)andintrauterinedevice(RR=1.02,0.36–2.87,p=0.97).

Conclusion:PTENseemsnottobeusefulaspredictivemarkerofresponsetotheconservativetreatmentof

EHandEC,regardlessoftheadministrationroute(oralorintrauterine)ofprogestins.Weadvisefuture

researchernottofurtherassessPTENasastand-alonepredictivemarker.

©2018ElsevierB.V.Allrightsreserved.

Contents

Introduction ... 105

Materialsandmethods ... 105

Studyprotocol ... 105

Searchstrategy ... 105

Studyselection ... 105

Riskofbiasassessment ... 105

Dataextraction ... 105

Dataanalysis ... 106

*Correspondingauthorat:GynecologyandObstetricsUnit,DepartmentofNeuroscience,ReproductiveSciencesandDentistry,SchoolofMedicine,UniversityofNaples FedericoII,ViaSergioPansini,5,Naples,80131,Italy.

E-mailaddress:anton.raffone@gmail.com(A.Raffone).

https://doi.org/10.1016/j.ejogrb.2018.10.025

0301-2115/©2018ElsevierB.V.Allrightsreserved.

ContentslistsavailableatScienceDirect

European

Journal

of

Obstetrics

&

Gynecology

and

Reproductive

Biology

Results ... 106

Selectionandcharacteristicsofthestudies ... 106

Riskofbiasassessment ... 106

PTENandtherapyresponse ... 106

Comment ... 106

Strengthsandlimitation ... 109

Conclusion ... 109

Disclosure ... 109

Financialsupport ... 109

References... 109

Introduction

Endometrialcanceristhe mostcommongynecologic malig-nancyintheWesternworld[1].Inover80%ofcases,itispreceded by endometrial hyperplasia (EH), an irregular proliferation of endometrialglands [2,3].Incidence ofEH is about132/100.000 amongwomen[4].

Therevised2014WHOclassificationrecognizestwotypesofEH based on the presence of cytologic atypia: EH without atypia (benign)andatypicalEH(premalignant)[3].The20-year-riskof progressionofEHwithoutatypiatocancerislessthan5%[5],thus itmaybemanagedwithobservationaloneandfollow-upbiopsies. Ontheotherhand,atypicalEHrequiresatotalhysterectomy,dueto ariskofprogressionof29%[5].Progestinsconstitutethetreatment ofchoiceforsymptomaticEHwithoutatypiaandforwomenwith atypicalEH whowishtopreserve theirfertilityor whoarenot suitableforsurgery[6].Progestinsmaystillbeusedfor fertility-sparing treatment of endometrioid type, stage FIGO IA, well-differentiated early endometrial cancer (EEC) without tumor invasion of myometrium [7]. Progestins can be administered orally or through intrauterine device (levonorgestrel-releasing intrauterinedevice,LNG-IUD)[6,8].

However,despiteprogestins’effectiveness,avariable percent-ageofEHandEECdonotrespondtotherapy,orevenprogressto invasivedisease[8].

Thishasledtoagrowinginterestinsearchingforpredictive markersofresponsetoprogestinsonendometrialspecimens,in order to avoid the risk of disease progression linked to an ineffectivetherapy.Inparticular,amajorrolehasbeenplayedby immunohistochemistry,whichisthemostwidelyusedtoolinthis field[9].

Thetumorsuppressorproteinphosphataseandtensinhomolog (PTEN)hasbeenoneofthemostimportantmarkersstudiedinthis field,duetoitsrecognizedroleinendometrialcarcinogenesis[2].A prognosticsignificanceofPTENexpressioninEHhasalreadybeen reportedfortheriskofconcurrentcancer[10]andprogressionto cancer [11]. Some authors also suggested a role of PTEN in resistancetoprogestins[12,13],althoughthis point hasnot yet beenclarified.

Thus,theaimofthissystematicreviewandmeta-analysiswas toassessifimmunohistochemicallossofPTENexpressionon pre-treatmentendometrialspecimenmaybea predictivemarkerof responsetoprogestinsinEHandEEC.

Materialsandmethods Studyprotocol

This study was performed according to a protocol recom-mended for systematic reviewand meta-analysis. The protocol definingmethodsforcollecting,extractingandanalyzingdatawas designedapriori.Allreviewstageswereconductedindependently bytworeviewers(ATandAR)anddisagreementswereresolvedby discussionwithathirdauthor(GS).

Searchstrategy

SeveralresearcheswereconductedusingMEDLINE,EMBASE, Web of Sciences, Scopus, ClinicalTrial.gov, OVID and Cochrane Library aselectronic databases. The studies wereidentified by using a combination of the following text words from the inception to May 2018: endometrial hyperplasia; endometrial cancer;endometrioidadenocarcinoma;endometrial intraepithe-lialneoplasia;EIN;therapy;treatment;fertilitysparing; conser-vative; medroxyprogesterone; MPA; mirena; LNG; levonorgestrel; progestogen; progestin; response; resistance; persistence; outcome;PTEN;phosphataseandtensinhomolog; marker;immunohistochemistry;immunohistochemical.Review ofarticlesalsoincludedtheabstractsofallreferencesretrieved fromthesearch.

Studyselection

Weincludedinoursystematicreviewallrandomizedand non-randomizedstudiesthatsatisfiedthefollowinginclusioncriteria: studypopulationconstitutedbywomendiagnosedwithEHor

EECandconservativelytreatedwithprogestogens;

assessment of the expression of PTEN on pre-treatment endometrialspecimensbyimmunohistochemistry;

assessment of the association between PTEN expression (presencevsloss) andtheresponsetotherapy(goodvspoor, where‘goodresponse’denotedacompleteregressionofEHor EEC);

comparabilityofdata. Riskofbiasassessment

According totheMethodological Index for Non-Randomized Studies(MINORS)[14],sixdomains relatedtoriskofbiaswere assessedineachstudy:1)Aim(i.e.clearlystatedaim),2)Rate(i.e. inclusion of consecutive patients), 3) Data (i.e. prospective collection of data), 4) Bias (i.e. unbiased assessment of study endpoints),5)Time(i.e.follow-uptimeappropriate),6)Loss(i.e. losstofollow-up).Reviewauthors’judgmentswerecategorizedas “lowrisk,”“highrisk”or“unclearriskofbias.”

Dataextraction

Data were extracted from each study without modification. Two by twocontingency tables were prepared reporting two dichotomous qualitative variables: PTEN expression on pre-treatmentbiopsy(“presence”vs“loss”)andresponseto conserva-tivetherapy(“good”vs“poor”,where“goodresponse”indicateda completeregressionofdisease).Whendiscrepanciesbetweentext andtableswerefound,valuesfromtableswereused.Inonestudy [15],2womentreatedwithLNG-IUDshowednoovertfindingsof EH on follow-up biopsy, although PTEN-null glands were still

present.Thesepatientswereconsideredaspoorresponders,since accordingtoMutteretalthepresenceofPTEN-nullglandswould indicatealatentprecancer[16]andthusnotacompleteregression. Data were also subdivided into subgroups based on the histologic diagnosis (EH or EEC) and administration route of progestins(oralorintrauterine).

Dataanalysis

TheimpactofPTENstatusonthetherapyoutcomewasassessed asrelativerisk (RR)forfailure of therapy,with95% confidence interval (CI). RR was calculated for each study and as pooled estimateandreportedgraphicallyonaforestplot.Ap-value<0.05 wasconsideredsignificant.

The inconsistencyindex(I2)was used fortheassessment of statistical heterogeneity among studies: heterogeneity was considered insignificant for I2_{<25%, low for I}2_{<50%, moderate}

forI2<75%andhighforI275%.IncaseofI250%arandomeffect model was used; in case of I2_{<50% a fixed effect model was}

adopted.

A subgroupanalysiswas alsoperformed(EHvsEEC;oral vs intrauterineprogestins).

Data analysis was performed using Review Manager 5.3 (Copenhagen:The NordicCochraneCentre,Cochrane Collabora-tion,2014).

Results

Selectionandcharacteristicsofthestudies

TheprocessofstudyselectionissummarizedinFig.1. Seven studies assessing 376 patients were included [12,13,15,17–20]. Five studies were retrospective and 2 were prospective.Thesamplesizerangedfrom9to141.

Outof376EH,217hadPTENlossand159hadPTENpresenceat immunohistochemistry.Patients with good responsewere 275, while101hadpoorresponse.Patientsagerangedfrom19to79.

PatientsBMIrangedfrom20to39.Histologicdiagnosisincluded 239non-atypicalEH,98atypicalEHand39EEC.

Progestinsusedincludedmegestrolacetate(N=65),norethindrone acetate(N=14),medroxyprogesteroneacetate(N=207)administered orally,LNG-IUD(N=84)oramixtureofmorethanoneprogestin(N=9). Follow-updurationrangedfrom1to26months.

CharacteristicsoftheincludedstudiesareshowninTable1. Riskofbiasassessment

Forthe“aim”domain,allstudieswerecategorizedatlowriskof bias,sincetheyhadaclearlystatedaim.

Forthe“rate”domain,threestudieswerecategorizedatlow risk, because they selected consecutive patients; the other 4 studiesdidnotclearlyspecifythispoint,althoughitseemsthat theyincludedalleligiblepatientsintheperiodconsidered,thus theywereconsideredatunclearrisk.

Forthe“data”domain,3studieswerecategorizedatlowrisk,since theycollected data prospectively; the other 4 studies were considered at unclearriskbecausethisinformationwasnotspecified.

For the “bias” domain, only one study was categorized at unclearrisk,becauseitconsiderednon-atypicalEHasaregression ofatypicalEH;theother6studieswereconsideredatlowrisk,asa diagnosiscompleteresponseimpliedthatnolesionshadpersisted atfollow-up.

Forthe“time”domain,4studieswerecategorizedatlowrisk, since they treated all patients at least for 6 months, as recommendedbyguidelines[6,7];fortheotherstudiestherisk wasunclearbecausethedurationoftherapywas1–6months.

Forthe“loss”domain,allstudieswerecategorizedatunclear risk, becausethenumber of patientslosttofollow-up was not specified.

ResultsofriskofbiasassessmentareshowninFig.2. PTENandtherapyresponse

AlossofPTENexpressionshoweda relativeriskfor therapy failureof1.24(95%CI,0.88–1.76);theimpactontheriskwasnot significant (p=0.21);therewas noheterogeneityamongstudies (I2_=0%)(Fig.3).

InthesubgroupofpatientswithEC(N=39),RRwas0.89(95% CI,0.32–2.49),withoutsignificantimpactonthetherapyoutcome (p=0.83)andwithoutheterogeneity(I2_{=0%).Inthesubgroupof}

patientswithEH(N=337),RRwas1.30(95%CI,0.90–1.87)without statisticalsignificance(p=0.16)andwithoutsignificant heteroge-neity(I2_{=16%).Therewasnosignificantdifference betweenthe}

twosubgroup(Chi2=0.45;p=0.50)(Fig.3).

On the basis of the treatment administered, the subgroup treatedwithoralprogestin(N=291)showedanon-significantRR of1.25(95%CI,0.88–1.79,p=0.22)withnoheterogeneity(I2_=0%).

InthesubgrouptreatedwithLNG-IUD(N=85),RRwas1.02(95%CI, 0.36–2.87)withoutstatisticalsignificance(p=0.97)andwithout heterogeneity(I2_{=0).Therewasnosignificantdifferencebetween}

thetwosubgroup(Chi2_{=0.14;p=0.71)(Fig.4).}

Comment

OurresultsshowedthatthelossofPTENexpression,evaluated at immunohistochemistry, does not affect the outcome of progestin-basedtherapyofEHandEC.

PTENgeneisatumor-suppressorgene,locatedatchromosome 10q23.Itencodesaproteinwithalipidphosphataseactivity,which inducescellcyclearrest,andfavorsapoptosisupregulating AKT-dependent mechanisms and downregulating Bcl-2-dependent mechanisms,actinginoppositiontoPI3K.PTENproducthasalso a proteinphosphataseactivity,involvedintheinhibitionof cell

Fig.1.Flowdiagramofstudiesidentifiedinthesystematicreview(Prismatemplate [PreferredReportingItemforSystematicReviewsandMeta-analyses]).

spread, focal adhesion formation and growth-factor-stimulated MAPKsignaling[21].

PTEN has played a major role in the study of endometrial carcinogenesis,sincePTENgeneisthemostcommonlymutatedin endometrioidcarcinoma[22].

In particular, PTEN hasbeenstudied asthe main marker of endometrial precancerous lesion; Mutteret al. indeedreported that PTEN loss at immunohistochemistry might differentiate premalignantEHfrombenignfunctionalEH[16,23].

In2013,theCancerGenomeAtlasResearchNetworkidentified fourdistinctmolecularcategoriesofEC.Inthecategories ‘ultra-mutated’,‘hypermutated’and‘copynumberlow’,whicharetypically endometrioid,PTENmutationswerefoundin94%,88%and77% respectively. In the last category (‘copy number high’), mainly constitutedbyserousEC,PTENmutationswerefoundonlyin15% ofspecimens[22].

A loss of PTEN expression was found to be prognostic for progressionofEHtoEC[11],andforthepresenceofacoexistentEC after a diagnosis of EH [10]. Given the widespread use of conservative therapy of EH and EC, great importance hasbeen given tothesearch forpredictive markersof response[24–27]. PTENhasstillplayeda majorrolein thisfield,althoughresults aboutitspredictiveabilityappearcontrasting[12,13,15,17–20].

T able 1 Charact eristics of the included studies. YEAR FIRS T A UTHOR CO UNTR Y S TUD Y DESIGN PERIOD OF ENR OLLMENT S A MPLE SIZE P A TIENTS ’ FEA TURES S AMPLIN G METHOD HIS TOLOG Y P R OGES TOGEN ADMINIS TERED TREA TMENT DURA TION RESPONSE A G E BMI N A H A H E C M G A NET A M P A LN G M IX GOOD POOR 20 0 7 Minaguchi Japan retr ospectiv e 1 989-20 03 3 1 1 9-60 n.r . cur ettag e – 12 1 9 –– 31 –– 2-1 8 months 26 5 20 0 7 Y a maza w a Japan pr ospectiv e 1 999-20 05 9 28-40 n.r . cur ettag e –– 9 –– 9 –– 6-9 months 7 2 20 08 Milam US A retr ospectiv e n.r . 3 8 20-79 n.r . biopsy 1 3 25 – 18 5 1 6 –– 1-1 2 months 1 6 22 20 1 0 Akesson UK pr ospectiv e 1 999-20 04 34 36-7 7 2 1-49 n.r . 2 9 5 –– – – 34 – 26 months 28 6 20 1 2 U pson US A retr ospectiv e 1 985-20 05 11 2 < 39 to > 70 < 25 to > 30 n.r . 7 2 4 0 – 46 9 5 0 – 9n s 8 0 3 2 20 1 5 Orbo N orw a y retr ospectiv e 2 0 05-20 11 1 4 1 < 44 to > 52 < 20 to > 30 Pipelle 1 2 5 1 6 –– – 93 48 – 6 months 11 2 2 9 20 1 6 Van Gent N etherla nds retr ospectiv e 2 0 02-20 1 2 11 2 7-38 20-39 biopsy , cur ettage –– 11 1 – 82 – 6-1 9 m onths 6 5 TOT AL 2 pro spectiv e 5 ret ro specti ve 1 985-20 1 2 3 7 6 1 9-79 20-39 – 239 98 39 65 1 4 20 7 8 4 9 1-26 months 2 7 5 1 0 1 N AH: non-atypical h yperplasia; AH: atypical h yperplasia; EC: endometrial cancer; MGA: meges trol acetat e; NET A: nor ethindrone acetate ; MP A: medr o xypro g est er one acetate ; LNG : levo norg estr el-re leasing intr aut erine device; MIX: mixtur e o f m ore than one pro g estin; n.r .: no t report ed.

Fig.2.Assessmentofriskofbias.Summaryofriskofbiasforeachstudy;Plussign: lowriskofbias;minussign:highriskofbias;questionmark:unclearriskofbias.

In patientswithEC,wefoundthatPTENlosswasassociated with good response to progestins (RR=0.89), although not significantly(p=0.83).Inthisregard,arecentstudyshowedthat PTEN loss at immunohistochemistry did not influence the prognosisinEC[29].

InthesubgroupofEH,PTENlossshowedaRRof1.30,which indicatesanegativeimpactontheresponsetotherapy,although stillwithoutstatisticalsignificance(p=0.16).SincePTENlossinEH was shown tobe predictive for therisk of cancer [10,11], it is possiblethataminorinfluenceontheresponsemayexist.

Evenwhen assessed separately in women treated withoral progestinsand LNG-IUD,thepredictive value ofPTEN losswas neversignificant.

Anyway,whilethestatisticalsignificancemightbeachievedby assessingalargersample,theimpactontheresponsetotherapy would stillappear insufficient tohaveabearing onthepatient management,duetothelowvaluesofRRobserved.Conservative managementofatypicalEH andEECis notindeedthestandard treatment,andtheriskofprogressiontoinvasivediseaseisalready takenintoaccount,sopatientsundergoaclosefollow-up.Being

Fig.3.PTENlossinpredictingtheresponsetotherapyinendometrialhyperplasiaandearlyendometrialcancer.Forestplotsreportgraphicallytherelativeriskfortherapy failureaccordingtothehistologicdiagnosis(endometrialhyperplasiavsendometrialcancer).

Fig.4.PTENlossinpredictingtheresponsetotherapyinendometrialhyperplasiaandearlyendometrialcancer.Forestplotsreportgraphicallytherelativeriskfortherapy failureaccordingtothetypeofprogestinadministered(oralvsintrauterine).

theregressionratesreportedforLNG-IUDover90%[8],apredictive markershouldincreasetheriskoffailureatleast3–4foldstohave aclinicalsignificance.

All these results indicate that PTEN immunohistochemical statusdoesnotsignificantlyimpactontheresponsetoprogestins inEHandEC.Thus,furtherstudiesinthisfieldshouldnomore assessPTENasastand-alonepredictivemarker.

Inthisregard,twostudiesamongthoseincludedinourreview suggestedapossiblepredictiveroleforPTENifassessedtogether withothermolecules.Inparticular,Minaguchietal.reportedthat thepresenceofatleastonebetweenPTENlossandlowexpression of phospho-AKT was associated with higher therapy failure, comparedtotheabsenceofboth[12].Milametal.showedthat adecreased PTENexpressionaccompaniedbya phospho-mTOR overexpressiononthefollow-upbiopsyisassociatedwithapoor response[13].Remarkably,bothphospho-AKTandphospho-mTOR are molecules involved in the same pathway as PTEN [12,13]. Nonetheless,evidenceinthisfielditisweakanditisunclearwhich significancethesefindingsmayhaveinthepatientmanagement. Severalotherpredictivemarkershavebeenassessedby immuno-histochemistry in EH and EC. Among these, estrogen and progesterone receptors have shown association with good responsetoprogestinsinsomestudies[17–19].Bycontrast,other studiesshowedoppositeresults[12,24],andEuropeanguidelines discourage their use [7]. One study showed that deficient expressionof mismatch repair proteins was predictive of poor response, with perfect specificity [27]; however, given some limitationsinherenttothestudypopulation,thisresultneedtobe furtherconfirmed[28].

Withtherecentprogressesaboutthemoleculardefinitionof endometrialneoplasticspecimens [22,29,30], inthenearfuture theroleofsuchmoleculesmightbeclarified.

Strengthsandlimitation

Tothebestofourknowledge,thisisthefirstreviewassessing theroleofPTENintheconservativemanagementofEHandEEC. OurstudyclarifiedthatimmunohistochemicalexpressionofPTEN doesnotaffecttheoutcomeofprogestin-therapyinEH andEC, independently from histologic category or type of progestin administered.

Alimitationofourstudymightbetherelativelysmallsizeofthe sampleassessed(N=376),althoughtheabsenceofheterogeneity among studies (I2=0%) and the constancy of RR values in the several groups give solidity to our results. Furthermore, we consideredPTENexpressionassessedbyonly immunohistochem-istry. Several concerns with PTEN immunohistochemistryhave been reported, such as subjectivity in the interpretation of immunostaining and lack of a standard protocol [31]. On the otherhand,immunohistochemistryallowsevaluatingboth inten-sityanddistributionofthemarkerexpression.Thereisevidence that PTEN immunohistochemistry may outperform PTEN gene sequencing[32]. For ouranalysis, we tookinto accountonly a completelossofimmunostaining,whichshouldbeeasilyreadable evenwithoutexpertiseinimmunohistochemistry.Anyway,tothe best of our knowledge, there are no studies evaluating the predictive valueofPTEN bytechniques otherthan immunohis-tochemistryinpatientswithEHand/orECtreatedwithprogestins progestin. Two studies assessed the association betweenPTEN mRNAandprogesteronereceptorexpression,showingconflicting results [33,34]. However, studies demonstrating associations betweenPTEN and progesterone receptorexpressionmayhave limitedvalue,astheexpressionofprogesteronereceptorseemsto notreliablyreflecttheresponsivenesstoprogestins[7].

Another limitation may be the absence of studies treating patients by hysteroscopic resection plus progestin, which has

recently been described as the most effective conservative treatmentforEHandEC[35,36].

Finally,wewereunabletoextractdataseparatelyforatypical andnon-atypicalEH.

Conclusion

LossofPTENexpressionseemsnottobeusefulaspredictive markerofresponsetoprogestinsinEHandEEC,regardlessofthe administration route (oral or intrauterine). We advise future researchernottofurtherassessPTENasastand-alonepredictive marker.However,thecombinedassessmentof PTENwithother markers mighthavea predictive utility,but furtherevidence is requiredinthisregard.

Disclosure

Theauthorsreportnoconflictofinterest. Financialsupport

Nofinancialsupportwasreceivedforthisstudy. References

[1]FerlayJ.,SoerjomataramI,DikshitR,etal.Cancerincidenceandmortality worldwide:sources,methodsandmajorpatternsinGLOBOCAN.IntJCancer 2012;2015(136):E359–86.

[2]ShermanME. Theoriesofendometrialcarcinogenesis: amultidisciplinary approach.ModPathol2000;13:295–308.

[3]KurmanRJ,CarcangiuML,HerringtonCS,YoungRH,editors.WHO classifica-tionoftumoursoffemalereproductiveorgans.4thed.Lyon:IARC;2014. [4]ReedSD,Newton KM,ClintonWL,EppleinM,GarciaR, AllisonK,etal.

Incidenceofendometrialhyperplasia.AmJObstetGynecol2009;200(678): e1–6.

[5]JrLaceyJV,ShermanME,RushBB,etal.Absoluteriskofendometrialcarcinoma during20-yearfollow-upamongwomenwithendometrialhyperplasia.JClin Oncol2010;28:788–92.

[6]ManagementofendometrialhyperplasiaGreen-topguidelineNo.67RCOG/ BSGEjointguideline|February.2016.

[7]Colombo,etal.ESMO-ESGO-ESTROconsensusConferenceonendometrial cancer:diagnosis,treatmentandfollow-up.AnnOncol2016;27:16–41. [8]ZhangQ,QiG,KanisMJ,etal.Comparisonamongfertility-sparingtherapies

for well differentiated early-stage endometrial carcinoma and complex atypicalhyperplasia.Oncotarget2017;8(34):57642–53.

[9]SandersonPA,CritchleyHOD,WilliamsARW,ArendsMJ,SaundersPTK.New conceptsforanoldproblem:thediagnosisofendometrialhyperplasia.Hum ReprodUpdate2017;23(2):232–54.

[10]PavlakisK,MessiniI,VrekoussisT,etal.PTEN-lossandnuclearatypiaofEINin endometrialbiopsiescanpredicttheexistenceofaconcurrentendometrial carcinoma.GynecolOncol2010;119(3):516–9.

[11]BaakJP, VanDiermenB, SteinbakkA, etal. Lack ofPTENexpression in endometrialintraepithelialneoplasiaiscorrelatedwithcancerprogression. HumPathol2005;36(5):555–61.

[12]MinaguchiT,NakagawaS,TakazawaY,etal.Combinedphospho-aktandPTEN expressionsassociatedwithpost-treatmenthysterectomyafterconservative progestin therapy in complex atypical hyperplasia and stage Ia, G1 adenocarcinomaoftheendometrium.CancerLett2007;248(1):112–22. [13]MilamMR,Soliman PT,ChungLH,etal.Loss ofphosphataseand tensin

homologuedeletedonchromosome10andphosphorylationofmammalian targetofrapamycinareassociatedwithprogesteronerefractoryendometrial hyperplasia.IntJGynecolCancer2008;18(1):146–51.

[14]SlimK,NiniE,ForestierD,KwiatkowskiF,PanisY,ChipponiJ.Methodological indexfornon-randomizedstudies(minors):developmentandvalidationofa newinstrument.ANZJSurg2003;73(9):712–6.

[15]OrboA,ArnesM,LysaLM,StraumeB.ExpressionofPAX2andPTENassociates to therapy response in endometrial hyperplasia. Anticancer Res 2015;35:6401–9.

[16]MutterGL,InceTA,BaakJPA,KustGA,ZhouXP,EngC.Molecularidentification of latent precancers in histologically Normal endometrium. Cancer Res 2001;61:4311–4.

[17]Yamazawa K,HiraiM,FujitoA,etal. Fertility-preservingtreatmentwith progestin,andpathologicalcriteriatopredictresponses,inyoungwomenwith endometrialcancer.HumReprod2007;22(7):1953–8.

[18]AkessonE,GallosID,GanesanR,VarmaR,GuptaJK.Prognosticsignificanceof estrogenandprogesteronereceptorexpressioninLNG-IUS(mirena)treatment of endometrialhyperplasia: an immunohistochemicalstudy. Acta Obstet GynecolScand2010;89(3):393–8.

[19]UpsonK,AllisonKH,ReedSD,etal.Biomarkersofprogestintherapyresistance andendometrialhyperplasiaprogression.AmJObstetGynecol2012;207(1) e1–836.

[20]vanGentMD,Nicolae-CristeaAR,deKroonCD,etal.Exploringmorphologic andmolecularaspectsofendometrialcancerunderprogesteronetreatmentin thecontextoffertilitypreservation.IntJGynecolCancer2016;26(3):483–90. [21]OkudaTsuyoshi,SekizawaAkihiko,PurwosunuYuditiya,etal.Geneticsof

endometrialcancers.ObstetGynecolInt2010;2010:984013.

[22]CancerGenomeAtlasResearchNetwork,etal.Integratedgenomic characteri-zationofendometrialcarcinoma.Nature2013;497(7447):67–73.

[23]MutterGL,LinMC,FitzgeraldJT,etal.AlteredPTENexpressionasadiagnostic markerfortheearliestendometrialprecancers.JNatlCancerInst2000;92 (11):924–30.

[24]GundersonCC,DuttaS,FaderAN,etal.Pathologicfeaturesassociatedwith resolution of complex atypical hyperplasia and grade 1 endometrial adenocarcinomaafterprogestintherapy.GynecolOncol2014;132(1):33–7. [25]ØrboA,ArnesM,LysåLM,BorgfeldtC,StraumeB.HE4isanoveltissuemarker

for therapy responseand progestin resistance inmedium-and low-risk endometrialhyperplasia.BrJCancer2016;115(6):725–30.

[26]TierneyKE,JiL,DrallaSS,etal.Endoplasmicreticulumstressincomplex atypicalhyperplasiaasapossiblepredictorofoccultcarcinomaandprogestin response.GynecolOncol2016;143(3):650–4.

[27]ZakhourM,CohenJG,GibsonA,etal.Abnormalmismatchrepairandother clinicopathologicpredictorsofpoorresponsetoprogestintreatmentinyoung womenwithendometrialcomplexatypicalhyperplasiaand well-differenti-atedendometrialadenocarcinoma:aconsecutivecaseseries.BJOG2017;124 (10):1576–83.

[28]DerbyshireAE,RyanN,CrosbieEJ.Biomarkersneededtopredictprogestin responseinendometrialcancer.BJOG2017;124(September10):1584.

[29]KarnezisAN,LeungS,MagrillJ,etal.Evaluationofendometrialcarcinoma prognostic immunohistochemistry markers in the context of molecular classification.JPatholClinRes2017;3(4):279–93.

[30]TravaglinoA,RaffoneA,SacconeG,etal.Lossofbcl-2immunohistochemical expressioninendometrialhyperplasia:aspecificmarkerofprecancerand novelindicationfortreatment.Asystematicreviewandmeta-analysis.Acta ObstetGynecolScand2018(Aug31).

[31]BinghamV,OngCW,JamesJ,etal.PTENmRNAdetectionbychromogenic,RNA insitutechnologies:areliablealternativetoPTENimmunohistochemistry. HumPathol2016;47(January1):95–103.

[32]DjordjevicB,HennessyBT,LiJ, etal.ClinicalassessmentofPTENloss in endometrialcarcinoma:immunohistochemistryOut-performsGene sequenc-ing.ModPathol2012;25(May5):699–708.

[33]Samulak D, Grosman-Dziewiszek P, Michalska MM, et al. Evaluation of expression of the PTEN gene, oestrogen and progesterone receptors as diagnosticandpredictivefactorsinendometrialcancer.PatholOncolRes 2014;20(January1):191–6.

[34]SkrzypczakM,MerxI,Schüler-ToprakS,etal.Molecularprofilingofestrogen receptorα and progesterone receptor transcriptvariants inendometrial cancer.Steroids2015;104:122–8Dec.

[35]ZhangQ,QiG,KanisMJ,etal.Comparisonamongfertility-sparingtherapies for well differentiated early-stage endometrial carcinoma and complex atypicalhyperplasia.Oncotarget2017;3;8(May34):57642–53.

[36]GiampaolinoP,DiSpiezioSardoA,MolloA,etal.Hysteroscopicendometrial focalresectionfollowedbylevonorgestrelintrauterinedeviceinsertionasa fertility-sparing treatment ofatypical endometrial hyperplasia and early endometrialcancer:aretrospectivestudy.JMinimInvasiveGynecol2018;11 (July18)30347–9pii:S1553-S4650.